CN110325646B - Process for preparing (3E, 7E) -Gao Fani acid or (3E, 7E) -Gao Fani acid esters - Google Patents
Process for preparing (3E, 7E) -Gao Fani acid or (3E, 7E) -Gao Fani acid esters Download PDFInfo
- Publication number
- CN110325646B CN110325646B CN201880013734.XA CN201880013734A CN110325646B CN 110325646 B CN110325646 B CN 110325646B CN 201880013734 A CN201880013734 A CN 201880013734A CN 110325646 B CN110325646 B CN 110325646B
- Authority
- CN
- China
- Prior art keywords
- acid
- fani
- gao
- unsaturated
- formula
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000002253 acid Substances 0.000 title claims abstract description 126
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 16
- 150000002148 esters Chemical class 0.000 title claims description 39
- 238000000034 method Methods 0.000 claims abstract description 114
- 239000000203 mixture Substances 0.000 claims abstract description 61
- 230000008569 process Effects 0.000 claims abstract description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 25
- YPZUZOLGGMJZJO-UHFFFAOYSA-N ambrofix Natural products C1CC2C(C)(C)CCCC2(C)C2C1(C)OCC2 YPZUZOLGGMJZJO-UHFFFAOYSA-N 0.000 claims abstract description 22
- YPZUZOLGGMJZJO-LQKXBSAESA-N ambroxan Chemical compound CC([C@@H]1CC2)(C)CCC[C@]1(C)[C@@H]1[C@]2(C)OCC1 YPZUZOLGGMJZJO-LQKXBSAESA-N 0.000 claims abstract description 20
- 238000002360 preparation method Methods 0.000 claims abstract description 17
- 150000001732 carboxylic acid derivatives Chemical class 0.000 claims abstract description 10
- -1 carboxylic acid compound Chemical class 0.000 claims description 83
- 238000006243 chemical reaction Methods 0.000 claims description 47
- 108090001060 Lipase Proteins 0.000 claims description 36
- 102000004882 Lipase Human genes 0.000 claims description 33
- 239000004367 Lipase Substances 0.000 claims description 32
- 235000019421 lipase Nutrition 0.000 claims description 32
- 101710095468 Cyclase Proteins 0.000 claims description 31
- 230000002255 enzymatic effect Effects 0.000 claims description 29
- 125000001183 hydrocarbyl group Chemical group 0.000 claims description 21
- 239000003960 organic solvent Substances 0.000 claims description 20
- 230000000694 effects Effects 0.000 claims description 19
- 229920006395 saturated elastomer Polymers 0.000 claims description 19
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 17
- 150000001875 compounds Chemical class 0.000 claims description 15
- 238000005886 esterification reaction Methods 0.000 claims description 14
- 239000002904 solvent Substances 0.000 claims description 14
- 239000011541 reaction mixture Substances 0.000 claims description 13
- IMKJGXCIJJXALX-SHUKQUCYSA-N Norambreinolide Chemical compound CC([C@@H]1CC2)(C)CCC[C@]1(C)[C@@H]1[C@]2(C)OC(=O)C1 IMKJGXCIJJXALX-SHUKQUCYSA-N 0.000 claims description 12
- IMKJGXCIJJXALX-UHFFFAOYSA-N ent-Norambreinolide Natural products C1CC2C(C)(C)CCCC2(C)C2C1(C)OC(=O)C2 IMKJGXCIJJXALX-UHFFFAOYSA-N 0.000 claims description 12
- 230000032050 esterification Effects 0.000 claims description 12
- 229940096995 sclareolide Drugs 0.000 claims description 12
- 229910052799 carbon Inorganic materials 0.000 claims description 11
- 241000588902 Zymomonas mobilis Species 0.000 claims description 10
- 238000007257 deesterification reaction Methods 0.000 claims description 10
- 125000004030 farnesyl group Chemical group [H]C([*])([H])C([H])=C(C([H])([H])[H])C([H])([H])C([H])([H])C([H])=C(C([H])([H])[H])C([H])([H])C([H])([H])C([H])=C(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 10
- 125000004432 carbon atom Chemical group C* 0.000 claims description 7
- 238000004821 distillation Methods 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 238000007363 ring formation reaction Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 5
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 claims description 5
- 229960005174 ambroxol Drugs 0.000 claims description 4
- 108090000992 Transferases Proteins 0.000 claims description 3
- 102000004357 Transferases Human genes 0.000 claims description 3
- 150000001733 carboxylic acid esters Chemical class 0.000 claims description 3
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 2
- 239000012084 conversion product Substances 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 102100031375 Endothelial lipase Human genes 0.000 claims 1
- 230000002210 biocatalytic effect Effects 0.000 abstract description 8
- CRDAMVZIKSXKFV-FBXUGWQNSA-N (2-cis,6-cis)-farnesol Chemical compound CC(C)=CCC\C(C)=C/CC\C(C)=C/CO CRDAMVZIKSXKFV-FBXUGWQNSA-N 0.000 abstract description 3
- 239000000260 (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol Substances 0.000 abstract description 3
- 229940043259 farnesol Drugs 0.000 abstract description 3
- 229930002886 farnesol Natural products 0.000 abstract description 3
- 238000009472 formulation Methods 0.000 abstract description 3
- 239000007858 starting material Substances 0.000 abstract description 3
- CRDAMVZIKSXKFV-UHFFFAOYSA-N trans-Farnesol Natural products CC(C)=CCCC(C)=CCCC(C)=CCO CRDAMVZIKSXKFV-UHFFFAOYSA-N 0.000 abstract description 3
- 150000007523 nucleic acids Chemical class 0.000 description 74
- 102000004190 Enzymes Human genes 0.000 description 55
- 108090000790 Enzymes Proteins 0.000 description 55
- 229940088598 enzyme Drugs 0.000 description 55
- 108020004707 nucleic acids Proteins 0.000 description 51
- 102000039446 nucleic acids Human genes 0.000 description 51
- 108090000623 proteins and genes Proteins 0.000 description 45
- 125000000217 alkyl group Chemical group 0.000 description 28
- 230000014509 gene expression Effects 0.000 description 26
- 235000019441 ethanol Nutrition 0.000 description 24
- 108020004414 DNA Proteins 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 22
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 21
- 239000013598 vector Substances 0.000 description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 20
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 18
- 238000009396 hybridization Methods 0.000 description 17
- 229920001184 polypeptide Polymers 0.000 description 17
- 108090000765 processed proteins & peptides Proteins 0.000 description 17
- 102000004196 processed proteins & peptides Human genes 0.000 description 17
- 239000000126 substance Substances 0.000 description 17
- 235000018102 proteins Nutrition 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 16
- 150000003839 salts Chemical group 0.000 description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 description 15
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 15
- 125000003729 nucleotide group Chemical group 0.000 description 15
- 239000000047 product Substances 0.000 description 15
- 230000001105 regulatory effect Effects 0.000 description 15
- 239000002609 medium Substances 0.000 description 14
- 239000002773 nucleotide Substances 0.000 description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- 241001661345 Moesziomyces antarcticus Species 0.000 description 12
- 239000000758 substrate Substances 0.000 description 12
- 244000005700 microbiome Species 0.000 description 11
- 108091034117 Oligonucleotide Proteins 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 150000001298 alcohols Chemical class 0.000 description 9
- 239000012634 fragment Substances 0.000 description 9
- 108010031797 Candida antarctica lipase B Proteins 0.000 description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 8
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 8
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 7
- 125000003342 alkenyl group Chemical group 0.000 description 7
- 239000000284 extract Substances 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 239000013615 primer Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- 108010084311 Novozyme 435 Proteins 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 150000007513 acids Chemical class 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 230000000295 complement effect Effects 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 238000004817 gas chromatography Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 5
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 5
- 230000033228 biological regulation Effects 0.000 description 5
- 150000001735 carboxylic acids Chemical class 0.000 description 5
- 239000012876 carrier material Substances 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 5
- 238000010369 molecular cloning Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 241000640374 Alicyclobacillus acidocaldarius Species 0.000 description 4
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 4
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 241000187654 Nocardia Species 0.000 description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 125000001931 aliphatic group Chemical group 0.000 description 4
- 125000000304 alkynyl group Chemical group 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 229930195733 hydrocarbon Natural products 0.000 description 4
- 150000004702 methyl esters Chemical class 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000001509 sodium citrate Substances 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 239000008096 xylene Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- 108700010070 Codon Usage Proteins 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- 101710098554 Lipase B Proteins 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 3
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 239000003599 detergent Substances 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 3
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 3
- 235000019626 lipase activity Nutrition 0.000 description 3
- 230000008018 melting Effects 0.000 description 3
- 238000002844 melting Methods 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 3
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- 235000005985 organic acids Nutrition 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 239000002987 primer (paints) Substances 0.000 description 3
- DCKVNWZUADLDEH-UHFFFAOYSA-N sec-butyl acetate Chemical compound CCC(C)OC(C)=O DCKVNWZUADLDEH-UHFFFAOYSA-N 0.000 description 3
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 3
- 238000004904 shortening Methods 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 238000002604 ultrasonography Methods 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- KOICZDDAVPYKKX-UHFFFAOYSA-N (3E,7Z)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol Chemical compound CC(=C/CCO)CCC=C(/CCC=C(C)C)C KOICZDDAVPYKKX-UHFFFAOYSA-N 0.000 description 2
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- BBMCTIGTTCKYKF-UHFFFAOYSA-N 1-heptanol Chemical compound CCCCCCCO BBMCTIGTTCKYKF-UHFFFAOYSA-N 0.000 description 2
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 2
- 241000589291 Acinetobacter Species 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000589174 Bradyrhizobium japonicum Species 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 239000004215 Carbon black (E152) Substances 0.000 description 2
- 241000186216 Corynebacterium Species 0.000 description 2
- 241000186226 Corynebacterium glutamicum Species 0.000 description 2
- RGSFGYAAUTVSQA-UHFFFAOYSA-N Cyclopentane Chemical compound C1CCCC1 RGSFGYAAUTVSQA-UHFFFAOYSA-N 0.000 description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical class S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- 241001337904 Gordonia <angiosperm> Species 0.000 description 2
- 102100031415 Hepatic triacylglycerol lipase Human genes 0.000 description 2
- 108090000856 Lyases Proteins 0.000 description 2
- 102000004317 Lyases Human genes 0.000 description 2
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 2
- 241000190950 Rhodopseudomonas palustris Species 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- 241000187432 Streptomyces coelicolor Species 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- UYXTWWCETRIEDR-UHFFFAOYSA-N Tributyrin Chemical compound CCCC(=O)OCC(OC(=O)CCC)COC(=O)CCC UYXTWWCETRIEDR-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 2
- 125000003545 alkoxy group Chemical group 0.000 description 2
- 125000004414 alkyl thio group Chemical group 0.000 description 2
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 238000010923 batch production Methods 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 238000011138 biotechnological process Methods 0.000 description 2
- BTANRVKWQNVYAZ-UHFFFAOYSA-N butan-2-ol Chemical compound CCC(C)O BTANRVKWQNVYAZ-UHFFFAOYSA-N 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- KBPLFHHGFOOTCA-UHFFFAOYSA-N caprylic alcohol Natural products CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 125000002843 carboxylic acid group Chemical group 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 description 2
- 238000012411 cloning technique Methods 0.000 description 2
- 238000002742 combinatorial mutagenesis Methods 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 150000002170 ethers Chemical class 0.000 description 2
- 125000004494 ethyl ester group Chemical group 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 125000003187 heptyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- ZSIAUFGUXNUGDI-UHFFFAOYSA-N hexan-1-ol Chemical compound CCCCCCO ZSIAUFGUXNUGDI-UHFFFAOYSA-N 0.000 description 2
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 150000002430 hydrocarbons Chemical class 0.000 description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 235000013379 molasses Nutrition 0.000 description 2
- 238000002887 multiple sequence alignment Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- IXQGCWUGDFDQMF-UHFFFAOYSA-N o-Hydroxyethylbenzene Natural products CCC1=CC=CC=C1O IXQGCWUGDFDQMF-UHFFFAOYSA-N 0.000 description 2
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 239000012429 reaction media Substances 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 238000006722 reduction reaction Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000007127 saponification reaction Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- NUMQCACRALPSHD-UHFFFAOYSA-N tert-butyl ethyl ether Chemical compound CCOC(C)(C)C NUMQCACRALPSHD-UHFFFAOYSA-N 0.000 description 2
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- NLEBIOOXCVAHBD-YHBSTRCHSA-N (2r,3r,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-dodecoxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](OCCCCCCCCCCCC)O[C@H](CO)[C@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 NLEBIOOXCVAHBD-YHBSTRCHSA-N 0.000 description 1
- YPZUZOLGGMJZJO-CLWVCHIJSA-N (3as,5ar,9ar,9bs)-3a,6,6,9a-tetramethyl-2,4,5,5a,7,8,9,9b-octahydro-1h-benzo[e][1]benzofuran Chemical compound CC([C@H]1CC2)(C)CCC[C@@]1(C)[C@H]1[C@@]2(C)OCC1 YPZUZOLGGMJZJO-CLWVCHIJSA-N 0.000 description 1
- FCINNSNPGOGNMQ-YFVJMOTDSA-N (3e,7e)-4,8,12-trimethyltrideca-3,7,11-trienoic acid Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC(O)=O FCINNSNPGOGNMQ-YFVJMOTDSA-N 0.000 description 1
- KOICZDDAVPYKKX-HDVIWIBHSA-N (3z,7e)-4,8,12-trimethyltrideca-3,7,11-trien-1-ol Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C/CCO KOICZDDAVPYKKX-HDVIWIBHSA-N 0.000 description 1
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 1
- 125000006659 (C1-C20) hydrocarbyl group Chemical group 0.000 description 1
- 125000005918 1,2-dimethylbutyl group Chemical group 0.000 description 1
- OCJBOOLMMGQPQU-UHFFFAOYSA-N 1,4-dichlorobenzene Chemical compound ClC1=CC=C(Cl)C=C1 OCJBOOLMMGQPQU-UHFFFAOYSA-N 0.000 description 1
- DURPTKYDGMDSBL-UHFFFAOYSA-N 1-butoxybutane Chemical compound CCCCOCCCC DURPTKYDGMDSBL-UHFFFAOYSA-N 0.000 description 1
- 125000006218 1-ethylbutyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C([H])([H])[H] 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- WIIZWVCIJKGZOK-IUCAKERBSA-N 2,2-dichloro-n-[(1s,2s)-1,3-dihydroxy-1-(4-nitrophenyl)propan-2-yl]acetamide Chemical compound ClC(Cl)C(=O)N[C@@H](CO)[C@@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-IUCAKERBSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 125000006176 2-ethylbutyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(C([H])([H])*)C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000004493 2-methylbut-1-yl group Chemical group CC(C*)CC 0.000 description 1
- 125000005916 2-methylpentyl group Chemical group 0.000 description 1
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 1
- 125000003542 3-methylbutan-2-yl group Chemical group [H]C([H])([H])C([H])(*)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000005917 3-methylpentyl group Chemical group 0.000 description 1
- ACZGCWSMSTYWDQ-UHFFFAOYSA-N 3h-1-benzofuran-2-one Chemical compound C1=CC=C2OC(=O)CC2=C1 ACZGCWSMSTYWDQ-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- 229920000178 Acrylic resin Polymers 0.000 description 1
- 239000004925 Acrylic resin Substances 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000611272 Alcanivorax Species 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 244000105975 Antidesma platyphyllum Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 125000002853 C1-C4 hydroxyalkyl group Chemical group 0.000 description 1
- 125000003358 C2-C20 alkenyl group Chemical group 0.000 description 1
- 241000283153 Cetacea Species 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000186031 Corynebacteriaceae Species 0.000 description 1
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- ZAQJHHRNXZUBTE-NQXXGFSBSA-N D-ribulose Chemical compound OC[C@@H](O)[C@@H](O)C(=O)CO ZAQJHHRNXZUBTE-NQXXGFSBSA-N 0.000 description 1
- ZAQJHHRNXZUBTE-UHFFFAOYSA-N D-threo-2-Pentulose Natural products OCC(O)C(O)C(=O)CO ZAQJHHRNXZUBTE-UHFFFAOYSA-N 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- QRLVDLBMBULFAL-UHFFFAOYSA-N Digitonin Natural products CC1CCC2(OC1)OC3C(O)C4C5CCC6CC(OC7OC(CO)C(OC8OC(CO)C(O)C(OC9OCC(O)C(O)C9OC%10OC(CO)C(O)C(OC%11OC(CO)C(O)C(O)C%11O)C%10O)C8O)C(O)C7O)C(O)CC6(C)C5CCC4(C)C3C2C QRLVDLBMBULFAL-UHFFFAOYSA-N 0.000 description 1
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000187809 Frankia Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 206010071602 Genetic polymorphism Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229910010082 LiAlH Inorganic materials 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000589345 Methylococcus Species 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000255969 Pieris brassicae Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000222180 Pseudozyma tsukubaensis Species 0.000 description 1
- 108010019653 Pwo polymerase Proteins 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 229910003798 SPO2 Inorganic materials 0.000 description 1
- 101100434411 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ADH1 gene Proteins 0.000 description 1
- 101100478210 Schizosaccharomyces pombe (strain 972 / ATCC 24843) spo2 gene Proteins 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 108010034546 Serratia marcescens nuclease Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 108091000048 Squalene hopene cyclase Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 241000204066 Tsukamurella Species 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- ZVNYJIZDIRKMBF-UHFFFAOYSA-N Vesnarinone Chemical compound C1=C(OC)C(OC)=CC=C1C(=O)N1CCN(C=2C=C3CCC(=O)NC3=CC=2)CC1 ZVNYJIZDIRKMBF-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 125000002015 acyclic group Chemical group 0.000 description 1
- 101150102866 adc1 gene Proteins 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000005599 alkyl carboxylate group Chemical group 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 239000012080 ambient air Substances 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000006229 amino acid addition Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000012501 ammonium carbonate Nutrition 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000012431 aqueous reaction media Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000001204 arachidyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 150000007514 bases Chemical class 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000008238 biochemical pathway Effects 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 230000002051 biphasic effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- CQEYYJKEWSMYFG-UHFFFAOYSA-N butyl acrylate Chemical compound CCCCOC(=O)C=C CQEYYJKEWSMYFG-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000000648 calcium alginate Substances 0.000 description 1
- 235000010410 calcium alginate Nutrition 0.000 description 1
- 229960002681 calcium alginate Drugs 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001721 carbon Chemical group 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 229940023913 cation exchange resins Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000004464 cereal grain Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000012707 chemical precursor Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000002734 clay mineral Substances 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- WJTCGQSWYFHTAC-UHFFFAOYSA-N cyclooctane Chemical compound C1CCCCCCC1 WJTCGQSWYFHTAC-UHFFFAOYSA-N 0.000 description 1
- 239000004914 cyclooctane Substances 0.000 description 1
- 125000002704 decyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 229940009976 deoxycholate Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 150000001983 dialkylethers Chemical class 0.000 description 1
- SHFGJEQAOUMGJM-UHFFFAOYSA-N dialuminum dipotassium disodium dioxosilane iron(3+) oxocalcium oxomagnesium oxygen(2-) Chemical compound [O--].[O--].[O--].[O--].[O--].[O--].[O--].[O--].[Na+].[Na+].[Al+3].[Al+3].[K+].[K+].[Fe+3].[Fe+3].O=[Mg].O=[Ca].O=[Si]=O SHFGJEQAOUMGJM-UHFFFAOYSA-N 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229940117389 dichlorobenzene Drugs 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- UVYVLBIGDKGWPX-KUAJCENISA-N digitonin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)C[C@@H](O)[C@H](O[C@H]5[C@@H]([C@@H](O)[C@@H](O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)CO7)O)[C@H](O)[C@@H](CO)O6)O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O7)O)[C@@H](O)[C@@H](CO)O6)O)[C@@H](CO)O5)O)C[C@@H]4CC[C@H]3[C@@H]2[C@@H]1O)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 UVYVLBIGDKGWPX-KUAJCENISA-N 0.000 description 1
- UVYVLBIGDKGWPX-UHFFFAOYSA-N digitonine Natural products CC1C(C2(CCC3C4(C)CC(O)C(OC5C(C(O)C(OC6C(C(OC7C(C(O)C(O)CO7)O)C(O)C(CO)O6)OC6C(C(OC7C(C(O)C(O)C(CO)O7)O)C(O)C(CO)O6)O)C(CO)O5)O)CC4CCC3C2C2O)C)C2OC11CCC(C)CO1 UVYVLBIGDKGWPX-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- POLCUAVZOMRGSN-UHFFFAOYSA-N dipropyl ether Chemical compound CCCOCCC POLCUAVZOMRGSN-UHFFFAOYSA-N 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000005187 foaming Methods 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000009760 functional impairment Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 235000009424 haa Nutrition 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- DMEGYFMYUHOHGS-UHFFFAOYSA-N heptamethylene Natural products C1CCCCCC1 DMEGYFMYUHOHGS-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 125000004491 isohexyl group Chemical group C(CCC(C)C)* 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- CYPPCCJJKNISFK-UHFFFAOYSA-J kaolinite Chemical compound [OH-].[OH-].[OH-].[OH-].[Al+3].[Al+3].[O-][Si](=O)O[Si]([O-])=O CYPPCCJJKNISFK-UHFFFAOYSA-J 0.000 description 1
- 229910052622 kaolinite Inorganic materials 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 101150109249 lacI gene Proteins 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 125000002960 margaryl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 125000001421 myristyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- 238000001320 near-infrared absorption spectroscopy Methods 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 125000001196 nonadecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 125000001400 nonyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 125000001117 oleyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 150000002898 organic sulfur compounds Chemical class 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000005022 packaging material Substances 0.000 description 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 125000002958 pentadecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000003538 pentan-3-yl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000010451 perlite Substances 0.000 description 1
- 235000019362 perlite Nutrition 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 229920001568 phenolic resin Polymers 0.000 description 1
- 239000005011 phenolic resin Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001522 polyglycol ester Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920000098 polyolefin Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 201000005484 prostate carcinoma in situ Diseases 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 235000008160 pyridoxine Nutrition 0.000 description 1
- 239000011677 pyridoxine Substances 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 125000003548 sec-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 230000037432 silent mutation Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000011537 solubilization buffer Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 229960000268 spectinomycin Drugs 0.000 description 1
- 238000012306 spectroscopic technique Methods 0.000 description 1
- 229940031439 squalene Drugs 0.000 description 1
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 1
- 108010088324 squalene cyclase Proteins 0.000 description 1
- 102000030633 squalene cyclase Human genes 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical class [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 150000004764 thiosulfuric acid derivatives Chemical class 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 125000002889 tridecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 125000002948 undecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
- C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/92—Naphthofurans; Hydrogenated naphthofurans
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y504/00—Intramolecular transferases (5.4)
- C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
- C12Y504/99017—Squalene--hopene cyclase (5.4.99.17)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention provides an improved process for separating the 3- (E) -isomer of an unsaturated carboxylic acid from a mixture of the corresponding (E/Z) isomers. More particularly, the present invention relates to an improved process for biocatalytically preparing (3E, 7E) -Gao Fani acid; and a novel biocatalytic process for the improved preparation of high farnesol, in particular (3E, 7E) -Gao Fani alcohol and (3E, 7E) -Gao Fani alcohol Gao Fani alcohol formulations with increased content. The invention also relates to a process for preparing (-) -ambrox by using (3E, 7E) -Gao Fani acid or (3E, 7E) -Gao Fani alcohol obtained according to the invention as starting material.
Description
The present invention provides an improved process for separating the 3- (E) -isomer of an unsaturated carboxylic acid from a corresponding (E/Z) isomer mixture. More particularly, the present invention relates to an improved process for biocatalytically preparing (3E, 7E) -Gao Fani acid (homofarnesylic acid); and a novel biocatalytic process for the improved preparation of high farnesol (homofarnesol), in particular (3E, 7E) -Gao Fani alcohol and high farnesol formulations with increased (3E, 7E) -Gao Fani alcohol (also referred to as "all-E-Gao Fani alcohol") content. The invention also relates to a process for preparing (-) -ambrox (ambrox) by using the (3E, 7E) -Gao Fani acid or (3E, 7E) -Gao Fani alcohol obtained according to the invention as starting material.
Background
Is enantiomerically pure compound (-) -ambroxol (3 aR,5aS,9 bR) -3a,6, 9 a-tetramethyl dodecahydronaphtho [2, 1-b)]Furan) which is used as a valuable fragrance. Naturally occurring (-) -ambrox is an ingredient of ambrox that is the digestion product of large white whales.
(-) -ambrox can be synthesized by applying chemical and/or enzymatic reaction steps (see scheme 1)
Scheme 1
The stereochemically pure forms of (3E, 7E) -Gao Fani acid or (3E, 7E) -Gao Fani alcohol are the most preferred starting materials for forming an enzyme-based synthetic route, as they allow the biosynthesis of (-) -ambrox with the correct stereochemistry.
A mixture of isomers (3 e,7 e) -and (3 z,7 e) -Gao Fani acid can be obtained. However, in view of the high similarity between the two isomers, it is difficult to separate such isomer mixtures. Thus, separation by classical methods such as distillation and chromatography is quite laborious.
It is therefore an object of the present invention to provide a process which allows to obtain stereoisomerically pure (3E, 7E) -Gao Fani acids and/or (3E, 7E) -Gao Fani alcohols more simply, or at least formulations with an increased content of (3E, 7E) -Gao Fani acids or (3E, 7E) -Gao Fani alcohols.
Brief description of the invention
Surprisingly, the above problems can be solved by providing an enzymatic catalytic process for the selective preparation of the 3E-isomer of unsaturated carboxylic acids, for example by the use of certain lipases (EC 3.1.1.3) catalysis, for the preparation of the 3E,7E isomer of Gao Fani acid.
Surprisingly, in a first particular aspect of the invention it has been found that by using the enzymatic activity of a lipase, for example a lipase from candida antarctica (Candida antarctica), the (3 e,7 e) -isomer of free Gao Fani acid is esterified more rapidly in the presence of an alcohol than the corresponding (3 z,7 e) -isomer. According to a first aspect of the invention, a mixture of (3E, 7E) -Gao Fani acid esters and free 3Z, 7E-Gao Fani acid is obtained. In view of the significant chemical differences between the acid and the ester, a very efficient and simple separation can now be performed by extraction or distillation. Chemical saponification of the thus isolated (3E, 7E) -Gao Fani acid ester yields the desired free 3E, 7E) -Gao Fani acid.
Surprisingly, it has also been found in a second particular aspect of the invention that by using the enzymatic activity of a lipase, for example a lipase from candida antarctica (Candida antarctica), the (3 e,7 e) -isomer of homofarnesyl esters saponifies more rapidly in the presence of water than the corresponding (3 z,7 e) -isomer. According to a second aspect of the invention, a mixture of (3E, 7E) -Gao Fani acid and 3Z, 7E-Gao Fani acid ester is obtained. In view of the significant chemical differences between the acid and the ester, a very efficient and simple separation can now be performed by extraction or distillation.
The following scheme 2 illustrates the two aspects of the invention:
scheme 2
Detailed Description
1. General definition:
without the opposite information, the following general definition should apply:
"Gao Fani acid (homofarnesylic acid)" or "homofarnesoic acid (homofarnesoic acid)" is a synonym for "(3E, 7E) -4,8, 12-trimethyltridec-3, 7, 11-trienoic acid" or "(3Z, 7E) -4,8, 12-trimethyltridec-3, 7, 11-trienoic acid" or a mixture of said E/Z isomers.
Sclareolide (Sclareolide) is used as a synonym for "(3 ar,5as,9 br) -3a,6, 9 a-tetramethyl-1, 4, 5a,7,8,9 b-octahydrobenzo [ e ] benzofuran-2-one".
The sclareolide shows the following structural formula:
"Ambrox" (Ambrox), and "Ambrox" (Ambrox) are used as synonyms. They include all stereoisomeric forms, for example, in particular (+) ambrox, 3 a-epi- (-) ambrox, 9 b-epi- (-) ambrox and in particular (-) ambrox.
According to the invention, the term "lipase" refers to a enzyme according to IUBMB enzyme nomenclature @http:// www.iubmb.unibe.ch;http://www.chem.qmul.ac.uk/iubmb/enzyme/) E.c.3.1.1.3 class of enzymes.
According to a particular embodiment of the method of the invention, the lipase is lipase B, a gene product of CALB from candida antarctica (Candida antarctica). CALB genes have been described previously (Uppenberg J., hansen, M.T., patkar, S., jones, A., structure 2:293-308 (1994)), and their nucleotide or protein sequences are saved under GenBank accession numbers Z30345 and CAA 83122.1. CALB herein means a nucleotide sequence having the search number unless specified more precisely. Another example of a triacylglycerol lipase is lipase B from Pseudozyma tsukubaensis (Suen, W.C., zhang, N., xiao, L, madison, V., zaks, A.protein Eng.Des.Sel.17 (2): 133-40 (2004)).
For the purposes of the present invention, a "cyclase" is generally an enzyme or enzyme mutant which in particular exhibits high farnesyl cyclase and/or high farnesyl cyclase activity. Enzymes having high farnesyl cyclase or high farnesyl cyclase activity are intramolecular transfer enzymes from a subset of isomerases; that is, a protein having EC number EC5.4 is suitable. (enzyme encoding according to Eur. J. Biochem.1999,264, 610-650). In particular, these are members of the EC 5.4.99.17 class. Suitable enzymes having high farnesyl cyclase or high farnesyl cyclase activity are in particular those which also achieve high farnesyl cyclization to sclareolide and/or squalene cyclization to graminene (hence sometimes also referred to as "SHC" or squalene-graminecyclase) and are described extensively in international patent application WO2010139719, which is incorporated herein by reference. Mutants thereof are for example described in WO2012/066059, which is expressly incorporated herein by reference.
The term "cyclase activity" describes an enzyme activity measured with a "reference substrate under standard conditions" which describes the formation of a cyclic product from an acyclic substrate. Standard conditions are, for example, a substrate concentration of 10mM to 0.2M, especially 15 to 100mM, for example about 20 to 25mM; at a pH of 4 to 8, the temperature is for example 15 to 30 or 20 to 25 ℃. Assays may be performed with cells expressing recombinant cyclase, cells expressing digested cyclase, fragments thereof or enriched or purified cyclase. Specifically, the reference substrate is (3E, 7E) -Gao Fani acid.
The "yield" and/or "conversion" of the reaction according to the invention is determined within a defined period, for example within 4, 6, 8, 10, 12, 16, 20, 24, 36 or 48 hours, during which the reaction takes place. In particular, the reaction is carried out under precisely defined conditions, for example at 25, 30, 40, 50 or 60 ℃.
An "enzyme catalyzed" or "biocatalytic" method refers to a method that is performed under the catalysis of an enzyme (including enzyme mutants), as defined herein. Thus, the method may be carried out in the presence of the enzyme in isolated (purified, enriched) or crude form or in the presence of a cellular system, in particular a natural or recombinant microbial cell containing the enzyme in active form, and has the ability to catalyze a conversion reaction as disclosed herein.
The term "selective conversion" or "increased selectivity" generally refers to a particular stereoisomeric form, such as the 3E-form of an unsaturated carboxylic acid as defined herein, which is converted in a higher proportion or amount (compared on a molar basis) than the corresponding 3Z-form throughout the reaction (i.e., between initiation and termination of the reaction), at some point in time of the reaction, or during the "intervals" of the reaction. In particular, the selectivity of conversion of the initial amount of substrate of the corresponding 1-99%, 2-95%, 3-90%, 5-85%, 10-80%, 15-75%, 20-70%, 25-65%,30-60% or 40-50% can be observed during the "interval". The higher proportion or amount may be expressed, for example, as follows:
The maximum yield of 3E-isomer observed during the whole reaction or during the intervals is higher;
-a higher relative amount of 3E-isomer at% of defined substrate conversion value; and/or
Equal relative amounts of 3E-isomer at% of higher conversion value;
each of which is preferably observed relative to a reference method that is performed chemically under otherwise identical conditions, such as chemical esterification or chemical ester cleavage.
The term "about" refers to a potential change of the values + -25%, especially + -15%, + -10%, preferably + -5%, + -2% or + -1%.
The term "substantially" describes a range of values from about 80 to 100%, for example from 85 to 99.9%, in particular from 90 to 99.9%, preferably from 95 to 99.9%, or from 98 to 99.9%, in particular from 99 to 99.9%.
"predominantly" means a proportion of more than 50%, for example 51-100%, preferably 75-99.9%; more particularly 85-98.5%, such as 95-99%.
Due to the reversibility of the enzymatic reaction, the present invention relates to the enzymatic reaction described herein in both reaction directions.
"functional mutants" of enzymes described herein include "functional equivalents" of such enzymes as defined below.
The term "stereoisomers" includes in particular conformational isomers.
According to the present invention, all stereoisomers, such as configurational isomers, particularly stereoisomers, and mixtures thereof, such as optical isomers, or geometric isomers, such as E and Z isomers, and combinations thereof, of the compounds are generally included herein. If several asymmetric centers are present in a molecule, the invention includes all combinations of different conformations of these asymmetric centers, e.g., pairs of enantiomers.
"stereoselectivity" describes the ability to produce a particular stereoisomer of a compound in stereoisomerically pure form or to specifically convert a particular stereoisomer from a plurality of stereoisomers by the enzyme-catalyzed processes described herein. More specifically, this means that the product of the invention is enriched in a particular stereoisomer. This can be quantified by the% purity ee parameter calculated according to the following formula:
%ee=[X A -X B ]/[X A +X B ]*100,
wherein X is A And X B Represents the molar ratio of stereoisomers A and B (Molenbroch).
"E-stereoselectivity" or "E-selectivity" describes the ability to produce at least one fold of an unsaturated E-isomer in pure or substantially pure or enriched form as the E-isomer on a particular c=c-double bond, or to convert the E-isomer at a particular position of the double bond specifically or substantially specifically from a plurality of other isomers or mixtures of E-and Z-isomers in the enzymatic methods described herein.
The definition of stereoselectivity above and its calculation are similarly applied to the term "enantioselectivity".
According to the invention, a stereoisomeric or enantiomeric purity of at least 90% ee, for example at least 95% ee, or at least 98% ee, or at least 99% ee or higher, may be obtained.
Unless otherwise indicated, the following general chemical definitions apply:
the term "carboxylic acid" includes the free acid and salt forms thereof, for example, their alkali or alkaline earth metal salts. This applies to all carboxylic acids mentioned herein, in particular Gao Fani acids.
The term "hydrocarbyl" group, linear or branched, saturated or unsaturated, according to the present invention, refers in particular to a linear or branched alkyl or alkenyl group.
The "alkyl" group comprising C 1 -C 20 Alkyl, which is a straight-chain or branched radical having 1 to 20 carbon atoms or C 1 -C 4 -alkyl or C 4 -C 20 -an alkyl group. Examples of which are:
-C 1 -C 4 an alkyl group selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl or tert-butyl,
-C 1 -C 6 -an alkyl group selected from C as defined above 1 -C 4 -alkyl groups, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-dimethylbutyl, 2, 3-dimethylbutyl, 3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1, 2-trimethylpropyl, 1, 2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl;
-C 7 -C 20 -an alkyl group, which is a linear or branched group having 7-20 carbon atoms; examples thereof are selected from the group consisting of heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl and structural isomers thereof, such as 4, 8-dimethylnonyl.
The "alkenyl" group comprises C 2 -C 20 Alkenyl which is mono-or polyunsaturated, in particular 1-, 2-, 3-or 4-fold, preferably 1-, 2-or 3-fold unsaturated, straight-chain or branched hydrocarbon radicals having from 2 to 20 carbon atoms.
Monounsaturated C 2 -C 20 Examples of alkenyl groups having a double bond in any position in the hydrocarbon chain are vinyl, 2-propen-1-yl, 1-methylpropan-2-en-1-yl, 2-buten-1-yl, 3-buten-1-yl, n-pentenyl,N-hexenyl, n-heptenyl, n-octenyl, n-nonenyl, n-decenyl, n-undecyl, n-dodecenyl, n-tridecenyl, n-tetradecenyl, n-pentadecenyl, n-hexadecenyl, n-heptadecenyl, n-octadecenyl, oleyl, n-nonadecenyl, eicosenyl;
di-or tri-unsaturated C having two or three double bonds, preferably non-cumulating and preferably non-conjugated bonds, at any position of the hydrocarbon chain 4 -C 20 Examples of alkenyl groups are n-butadienyl, n-pentadienyl, n-hexadienyl, n-heptadienyl, n-octadienyl, n-octatrienyl, n-nonadienyl, n-decadienyl, n-decatrienyl, n-undecadienyl, n-undecatrienyl, n-dodecadienyl, n-dodecatrienyl, n-tridecenyl, n-tetradecadienyl, n-tetradecatrienyl, n-pentadecenyl, n-hexadecadienyl, n-hexadecatrienyl, n-heptadecadienyl, n-octadecadienyl, n-octadecatrienyl, n-nonadienyl, n-nonadecatrienyl, n-icosatrienyl, and structural isomers thereof, such as 4, 8-dimethylnonen-3, 7-dienyl.
Unless otherwise indicated, C is as defined above 2 -C 20 Each double bond within an alkenyl group may take the E-or Z-configuration and, in the case of polyunsaturated, may be independently applicable to other double bonds.
Non-limiting examples of "optionally substituted" groups as defined herein include 1, 2, 3, 4, 5 or 6, preferably 1 or 2, identical or different substituents, such as HO, SH, NH 2 、NO 2 Halogen, such as F, cl, br, I; lower alkyl, lower alkoxy, lower alkylthio, lower alkyl, lower alkenyl, lower alkynyl or hydroxy-lower alkyl as defined above.
"lower alkyl" means C as defined above 1 -C 4 -an alkyl group.
"lower alkoxy" preferably refers to C of the lower alkyl groups mentioned above 1 -C 4 -alkoxy analogs.
"lower alkylthio" preferably refers to C of the lower alkyl groups mentioned above 1 -C 4 Alkylthio analogues. Examples are methylthio, ethylthio, propylthio, isopropylthio, butylthio, sec-butylthio, isobutylthio and tert-butylthio.
"lower alkenyl" includes C as defined above 2 -C 4 -alkenyl groups.
"lower alkynyl" includes alkynyl analogs of the "lower alkynyl" groups described above.
The term "hydroxy lower alkyl" means C 1 -C 4 Hydroxyalkyl, which is a linear or branched alkyl group having 1 to 4 carbon atoms, wherein at least one hydrogen atom, for example 1 or 2 hydrogen atoms, is substituted by a hydroxyl group. Examples thereof are hydroxymethyl, 2-hydroxy-1-ethyl, 2-and 3-hydroxy-1-propyl, 2-, 3-and 4-hydroxy-1-butyl, and structural isomers thereof.
Parameter ranges for different preferences of specific parameters are disclosed herein. Within the scope herein, any combination of ranges of parameters of different preferences for any combination of two or more parameters mentioned herein is also contemplated.
2. Specific embodiments of the invention:
the invention provides the following specific embodiments:
1. a process for separating the 3- (E) -isomer of an unsaturated carboxylic acid compound of the general formula (I) from an isomer mixture comprising the 3- (E) -and 3- (Z) -isomers of the carboxylic acid compound, wherein the isomer mixture is subjected to an enzymatic conversion reaction catalyzed by a lipase (EC 3.1.1.3), which preferably, in particular, stereoselectively converts the 3- (E) -isomer, and the conversion product of the 3- (E) -isomer is separated from the reaction mixture,
wherein the method comprises the steps of
R 1 Is H or straight or branched, saturatedOr unsaturated, optionally substituted C 1 -C 20 Hydrocarbyl groups, preferably saturated, unsubstituted, straight chain C 1 -C 20 A hydrocarbyl group;
R 3 is H or C 1 -C 4 -a hydrocarbon group, preferably H or methyl;
R 2 is a straight-chain or branched, saturated or unsaturated, optionally substituted C 1 -C 20 -a hydrocarbon group, preferably an unsaturated branched C 2 -C 16 -a hydrocarbon group;
provided that if R 3 Is C 1 -C 4 -a hydrocarbon group, R 2 Represents a hydrocarbon group containing at least one additional carbon atom.
2. The method of embodiment 1, wherein the lipase catalyzes a 3- (E) -stereoselective conversion reaction.
3. The method of any one of the preceding embodiments, wherein the lipase is applied in free or immobilized form.
4. The method of any one of the preceding embodiments, wherein the lipase is a naturally occurring or recombinantly produced optionally genetically modified (mutated) enzyme.
5. The method of any one of the preceding embodiments, wherein the lipase is from Candida sp.
6. The method of embodiment 5, wherein the lipase is Candida Antarctica Lipase B (CALB) comprising the amino acid sequence of SEQ ID No. 330 or a mutant thereof having at least 60% sequence identity with SEQ ID No. 330 and retaining the 3- (E) -selectivity.
7. The process according to any of the preceding embodiments, wherein the conversion reaction comprises an enzymatic esterification of an acid of formula (Ia), optionally in the presence of an organic solvent which does not interfere with or even inhibit lipase activity,
wherein the method comprises the steps of
R 2 And R is 3 As defined above;
and wherein predominantly 3- (E) -esters are formed; and wherein the aliphatic alkanol R 1 OH, wherein R is 1 As defined above, preference is given to straight-chain or branched, saturated C 1 -C 20 Alkyl, preferably C 1 -C 4 An alkyl group.
8. The method of any one of embodiments 1-6, wherein the conversion reaction comprises an enzymatic ester cleavage reaction of an ester of formula (Ib);
Wherein the method comprises the steps of
R 1 Is H or straight-chain or branched, saturated or unsaturated C 1 -C 20 -, in particular C 4 -C 20 -a hydrocarbon group;
and R is 2 And R is 3 As defined above;
and wherein predominantly 3- (E) -acid is formed; and wherein the reaction is carried out in the presence of water, optionally in the presence of an organic solvent which does not interfere with or even inhibit lipase activity
9. The process of any one of the preceding embodiments, wherein the conversion reaction is carried out in an organic solvent or a water-organic solvent.
10. The method of embodiment 9, wherein the organic solvent is selected from aliphatic or cycloaliphatic hydrocarbons, such as hydrocarbons having at least 5 carbon atoms, in particular hexane, cyclohexane, heptane, octane; aromatic hydrocarbons, in particular mononuclear aromatic compounds, such as benzene, xylene and toluene; and aliphatic ethers such as MTBE, diisopropyl ether. Preferably, a suitable organic solvent should be capable of forming a two-phase system with water.
11. The method of any one of the preceding embodiments, wherein the carboxylic acid compound is a 3- (E)/7- (E) -Gao Fani acid compound of formula (II),
wherein R is 1 As defined above.
12. A process for preparing an unsaturated 3- (E) -carboxylic acid of the general formula (Ia):
wherein the method comprises the steps of
R 2 And R is 3 As defined above;
wherein the method comprises the steps of
a) The isomer mixture of 3- (E) -and 3- (Z) -isomers comprising the carboxylic acid of formula (Ia) is represented by formula R 1 Enzymatic esterification in the presence of an alkanol of OH, wherein R is 1 C being linear or branched, saturated or unsaturated 1 -C 20 -a hydrocarbon group, preferably a linear or branched, saturated C 1 -C 20 Alkyl, preferably C 1 -C 4 An alkyl group; and in the presence of a lipase as defined in one of embodiments 1 to 6;
b) Separating the 3- (E) -carboxylic acid ester formed in step a), for example by transferring the unreacted acid isomer in the aqueous phase (as acid salt) and recovering the ester by an organic solvent which is present during the conversion or added after the conversion; and is also provided with
c) Saponifying the isolated ester of step b) to the corresponding 3- (E) -carboxylic acid of formula (Ia).
13. A process for preparing an unsaturated 3- (E) -carboxylic acid of the general formula (Ia):
wherein,
R 2 and R is 3 As defined above;
wherein the method comprises the steps of
a) A mixture of isomers comprising the 3- (E) -and 3- (Z) -isomers of the carboxylic acid esters of formula (Ib) is subjected to an enzymatic ester cleavage in the presence of a lipase as defined in one of embodiments 1 to 6, preferably in the presence of water and optionally an organic solvent,
wherein the method comprises the steps of
R 1 Is straight-chain or branched, saturated or unsaturated C 1 -C 20 Hydrocarbon radicals, preferably linear or branched, saturated C 1 -C 20 Alkyl, preferably C 1 -C 4 An alkyl group; and is also provided with
R 2 And R is 3 As defined above;
b) The 3- (E) -carboxylic acid formed in step a) is isolated, preferably as the free acid, from the aqueous phase of the reaction mixture.
14. The method according to embodiment 12 or 13, wherein an organic solvent as defined in any one of embodiments 9 and 10 is applied.
15. The method of any of the preceding embodiments, wherein the 3- (E) -isomer of the unsaturated carboxylic acid is 3- (E)/7- (E) -Gao Fani acid.
16. The method of any of the preceding embodiments, wherein the isomer mixture comprises a mixture of 3- (E)/7- (E) -Gao Fani acid and 3- (Z)/7- (E) -Gao Fani acid; or R is 1 Mixtures of 3- (E)/7- (E) -Gao Fani acid esters and 3- (Z)/7- (E) -Gao Fani acid esters of alkanols of OH, wherein R 1 Is straight-chain or branched, saturated or unsaturated C 1 -C 20 -a hydrocarbon group.
17. The process for preparing a 3- (E)/7- (E) -Gao Fani acid as in embodiment 16,
wherein the method comprises the steps of
a) The isomer mixture comprising 3- (E)/7- (E) -Gao Fani acid and 3- (Z)/7- (E) -Gao Fani acid is represented by formula R 1 Enzymatic esterification in the presence of an alkanol of OH, wherein R is 1 C being linear or branched, saturated or unsaturated 1 -C 20 -hydrocarbyl groupsPreferably straight-chain or branched, saturated C 1 -C 20 Alkyl, preferably C 1 -C 4 An alkyl group; and in the presence of a lipase as defined in any of embodiments 1 to 6 in a solvent as defined in one of embodiments 9 and 10;
b) Separating the 3- (E)/7- (E) -Gao Fani acid ester formed in step a) from unreacted acid (i.e., 3- (Z)/7- (E) -Gao Fani acid), particularly by distillation or preferably extraction, and
c) The isolated 3- (E)/7- (E) -Gao Fani acid ester was saponified to 3- (E)/7- (E) -Gao Fani acid.
18. The process for preparing 3- (E)/7- (E) -Gao Fani acid described in embodiment 16,
wherein the method comprises the steps of
a) Subjecting an isomer mixture comprising 3- (E)/7- (E) -Gao Fani acid ester and 3- (Z)/7- (E) -Gao Fani acid ester to an enzymatic ester cleavage reaction in the presence of a lipase as defined in one of embodiments 1-6 in a solvent as defined in one of embodiments 9 and 10, preferably in the presence of water; and is also provided with
b) The 3- (E)/7- (E) -Gao Fani acid formed in step a) is separated from the unreacted ester (i.e., 3- (Z)/7- (E) -Gao Fani acid ester), in particular by distillation or preferably extraction.
19. Preparation method of (-) -ambrox of formula (III)
The method comprises the following steps:
a) Obtaining the 3- (E)/7- (E) -Gao Fani acid by applying the method defined in any of embodiments 1-18;
b) Acid reduction of 3- (E)/7- (E) -Gao Fani to 3- (E)/7- (E) -Gao Fani alcohol and
c) The 3- (E)/7- (E) -Gao Fani alcohol is enzymatically cyclized to (-) -ambrox.
20. Preparation method of (-) -ambrox of formula (III)
The method comprises the following steps:
a) Obtaining said 3- (E)/7- (E) -Gao Fani acid by applying the method defined in any of embodiments 1-18;
b) Enzymatic cyclization of 3- (E)/7- (E) -Gao Fani acid to sclareolide of formula (IV),
and is also provided with
c) Chemically converting the sclareolide to (-) -ambroxol.
21. The method as in embodiment 19 or 20, wherein the enzymatic cyclization is carried out in the presence of an intramolecular transferase (e.c. 5.4), in particular a Gao Fani acid cyclase (e.c. 5.4.99.17), in particular a squalene-cereal cyclase (e.c. 5.4.99.17) exhibiting high farnesyl cyclase activity.
22. The method of embodiment 21, wherein the cyclase is a naturally occurring or recombinantly produced optionally genetically modified (mutated) squalene-cereal cyclase (SHC).
23. The method of embodiment 22, wherein the cyclase is squalene cyclase from methyl coccus capsulatus (Methylococcus capsalatus), rhodopseudomonas palustris (Rhodopseudomonas palustris), rhizobium japonicum (Bradyrhizobium japonicum), frank species (Frankia spec), streptomyces coelicolor (Streptomyces coelicolor) or, preferably, zymomonas mobilis (Zymomonas mobilis).
24. The method of embodiment 23, wherein the cyclase is from zymomonas mobilis (Zymomonas mobilis) comprising an amino acid sequence according to SEQ ID No. 2 or an amino acid sequence having at least 60% sequence identity to SEQ ID No. 2.
Alkyl 3- (E)/7- (E) -Gao Fani acid esters wherein the alkyl group is selected from C 2 -C 10 In particular C 3 -C 8 -alkyl groups such as ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl or tert-butyl; and pentyl, hexyl, heptyl, octyl and its structural isomers; preferably in a substantially stereoisomerically pure 3- (E)/7- (E) -form, and in particular in a form prepared by a process as defined above.
Typically, a cyclase suitable for use in the described aspects of the invention is SHC, which is listed below by reference to its wild-type sequence (SEQ ID NO and Genbank numbering) and its microbial origin.
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
/>
SEQ ID NO. 2 is the amino acid sequence of a cyclase also known as Zm-SHC-1.
3. The enzyme and enzyme mutant of the invention
The invention is not limited to the specifically disclosed uses of lipases and cyclases, but extends to functional equivalents thereof.
Within the scope of the present invention, the "functional equivalents" or analogues of the specifically disclosed enzymes are the various polypeptides thereof, which also have the desired biological function or activity, e.g. enzymatic activity.
For example, "functional equivalent" refers to an enzyme that exhibits at least 1-10%, or at least 20%, or at least 50%, or at least 75%, or at least 90% higher or lower enzyme activity as defined herein in a test for enzyme activity.
According to the invention, "functional equivalent" also refers in particular to a mutant which has an amino acid different from the specifically stated amino acid at least one sequence position of the above-mentioned amino acid sequences, but which nevertheless has one of the above-mentioned biological activities. Thus, "functional equivalents" comprise mutants obtainable by one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid additions, substitutions, insertions, deletions and/or inversions, wherein the changes may occur at any sequence position, provided that they result in mutants having the characteristic features of the invention. Functional equivalence is also provided in particular if the reactivity patterns qualitatively coincide between mutant and unchanged polypeptide, i.e. for example if the same substrate is converted at different rates. Examples of suitable amino acid substitutions are shown in the following table:
"functional equivalents" in the above sense are also "precursors" of the polypeptides, as well as "functional derivatives" and "salts" of the polypeptides.
In this case, a "precursor" is a natural or synthetic precursor of a polypeptide with or without the desired biological activity.
The expression "salt" refers to a salt of a carboxyl group of a protein molecule of the invention and an acid addition salt of an amino group. Salts of the carboxyl groups may be formed in known manner and include inorganic salts, such as sodium, calcium, ammonium, ferric and zinc salts, as well as salts with organic bases, such as amines, e.g., triethanolamine, arginine, lysine, piperidine, and the like. Acid addition salts, for example salts with inorganic acids such as hydrochloric acid or sulfuric acid and salts with organic acids such as acetic acid and oxalic acid, are also included in the present invention.
"functional derivatives" of the polypeptides according to the invention can also be produced on functional amino acid side chain groups or at their N-terminus or C-terminus using known techniques. These derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or primary or secondary amines; n-acyl derivatives of free amino groups produced by reaction with acyl groups; or O-acyl derivatives of free hydroxyl groups produced by reaction with acyl groups.
"functional equivalents" naturally also include polypeptides which can be obtained from other organisms, as well as naturally occurring variants. For example, regions of homologous sequence regions may be established by sequence comparison, and equivalent enzymes may be determined based on specific parameters of the invention.
"functional equivalents" also comprise fragments of the polypeptides of the invention, preferably individual domains or sequence motifs, which, for example, display the desired biological function.
Furthermore, a "functional equivalent" is a fusion protein having one of the above polypeptide sequences or a functional equivalent derived therefrom, and at least one other functionally different heterologous sequence associated at the functional N-terminus or C-terminus (i.e., without substantial mutual functional impairment of the fusion protein portions). Non-limiting examples of such heterologous sequences are, for example, signal peptides, histidine anchors or enzymes.
"functional equivalents" are also encompassed by the invention as homologs of the specifically disclosed proteins. They have the percent identity values described above. The values refer to identity to a specifically disclosed amino acid sequence and can be calculated according to the algorithm of Pearson and Lipman, proc.Natl.Acad, sci. (USA) 85 (8), 1988, 2444-2448.
The% identity value can also be calculated from BLAST alignment, algorithm blastp (protein-protein BLAST) or by applying the Clustal setting given below.
The percent identity of the homologous polypeptides of the present invention refers in particular to the percent identity of the amino acid residues relative to the total length of one of the amino acid sequences specifically described herein.
In the case of possible protein glycosylation, "functional equivalents" of the invention include proteins of the type described above in deglycosylated or glycosylated form as well as modified forms which can be obtained by altering the glycosylation pattern.
These functional equivalents or homologues of the proteins or polypeptides of the invention may be produced by mutagenesis, for example by point mutation, lengthening or shortening of the protein.
These functional equivalents or homologues of the proteins of the invention may be identified by screening combinatorial databases of mutants, e.g. shortening mutants. For example, a diverse database of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, e.g., by enzymatically ligating a mixture of synthetic oligonucleotides. There are a number of methods available for generating databases of potential homologs from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automated DNA synthesizer and the synthetic gene can then be ligated into a suitable expression vector. The use of a degenerate genome makes it possible to provide all sequences in a mixture which encode a desired set of potential protein sequences. Methods for synthesizing degenerate oligonucleotides are known to those skilled in the art (e.g., narag, S.A. (1983) Tetrahedron 39:3; itakura et al (1984) Annu. Rev. Biochem.53:323; itakura et al (1984) Science 198:1056; ike et al (1983) Nucleic Acids Res. 11:477).
In the prior art, several techniques are known for screening gene products of combinatorial databases generated by point mutation or shortening and for screening cDNA libraries of gene products having selected properties. These techniques may be suitable for rapid screening of gene libraries generated by combinatorial mutagenesis of homologs according to the invention. The most commonly used technique for screening large gene libraries is based on high throughput analysis, involving cloning the gene library in replicable expression vectors, transforming appropriate cells with the resulting vector database, and expressing the combined genes under conditions that will facilitate isolation of the vector encoding the gene whose product is being tested for the desired activity. Recursive ensemble mutagenesis (Recursive Ensemble Mutagenesis, REM) is a technique that increases the frequency of functional mutants in databases, and can be used in conjunction with these screening assays to identify homologs (Arkin and Yourvan (1992) PNAS 89:7811-7815; delgrave et al (1993) Protein Engineering 6 (3): 327-331).
4. Coding nucleic acid sequences
The invention also relates to nucleic acid sequences encoding the enzymes and mutants defined herein.
The present invention also relates to nucleic acids having a degree of "identity" to the sequences specifically disclosed herein. "identity" between two nucleic acids refers to the identity of the nucleotides, in each case over the entire length of the nucleic acid.
For example, it can be calculated by the Vector NTI Suite 7.1 program of Informax (USA) company using the Clustal method (Higgins DG, sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput appl. Biosci.1989 Apr;5 (2): 151-1) with the following settings:
multiple comparison parameters:
contrast parameters:
alternatively, identity may be determined by Chenna, ramu, sugawara, hideaki, koike, tadashi, lopez, rodrigo, gibson, toby J, higgins, desmond G, thompson, julie D.multiple sequence alignment with the Clustal series of programs (2003) Nucleic Acids Res (13): 3497-500, web pagehttp://www.ebi.ac.uk/Tools/clustalw/index.html#The method and the following settings were used for the determination:
all nucleic acid sequences mentioned herein (single-and double-stranded DNA and RNA sequences, such as cDNA and mRNA) can be produced in a known manner from nucleotide building blocks by chemical synthesis, for example by fragment condensation of individual overlapping complementary nucleic acid building blocks of a double helix. Chemical synthesis of oligonucleotides can be carried out in a known manner, for example by phosphoramidite method (Voet, voet, 2 nd edition, wiley Press, new York, pages 896-897). Synthetic oligonucleotides are accumulated and gaps filled by Klenow fragments of DNA polymerase and ligation reactions and general cloning techniques, which are described in Sambrook et al (1989), see below.
The invention also relates to nucleic acid sequences (single-and double-stranded DNA and RNA sequences, such as cDNA and mRNA) encoding one of the polypeptides described above and functional equivalents thereof, which can be obtained, for example, using artificial nucleotide analogs.
The present invention relates not only to isolated nucleic acid molecules which encode a polypeptide or protein of the invention or a biologically active segment thereof, but also to nucleic acid fragments which can be used, for example, as hybridization probes or primers for the identification or amplification of nucleic acids encoding the invention.
The nucleic acid molecules of the invention may additionally contain untranslated sequences from the 3 'and/or 5' end of the coding genetic region.
The invention also relates to nucleic acid molecules complementary to the specifically described nucleotide sequences or fragments thereof.
The nucleotide sequences of the present invention enable the preparation of probes and primers that can be used to identify and/or clone homologous sequences in other cell types and organisms. Such probes or primers typically comprise a region of nucleotide sequence that hybridizes under "stringent" conditions (see below) to at least about 12, preferably at least about 25, e.g., about 40, 50, or 75, consecutive nucleotides of the sense strand or the corresponding antisense strand of a nucleic acid sequence of the invention.
An "isolated" nucleic acid molecule is one that is isolated from other nucleic acid molecules that are present in the natural source of the nucleic acid and may be substantially free of other cellular material or culture medium (if it is produced by recombinant techniques), or may be free of chemical precursors or other chemicals (if it is synthesized chemically).
Nucleic acid molecules of the invention can be isolated by standard techniques of molecular biology and the sequence information provided by the invention. For example, cDNA may be isolated from a suitable cDNA library using one of the specifically disclosed complete sequences or fragments thereof as hybridization probes and standard hybridization techniques (e.g., as described in Sambrook, J., fritsch, E.F., and Maniatis, T.molecular Cloning: A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y., 1989). Alternatively, a nucleic acid molecule comprising one of the disclosed sequences or a fragment thereof may be isolated by polymerase chain reaction using oligonucleotide primers constructed based on the sequences. The nucleic acid amplified in this way can be cloned into a suitable vector and can be characterized by DNA sequencing. The oligonucleotides of the invention may also be produced by standard synthetic methods, for example using an automated DNA synthesizer.
The nucleic acid sequences of the invention or derivatives, homologues or parts of these sequences may be isolated from other bacteria, for example by conventional hybridization techniques or PCR techniques, for example by genomic or cDNA libraries. These DNA sequences hybridize to the sequences of the invention under standard conditions.
"hybridization" refers to the ability of a polynucleotide or oligonucleotide to bind to a nearly complementary sequence under standard conditions, without non-specific binding between non-complementary partners under these conditions. For this purpose, the sequence may be 90-100% complementary. The nature of the complementary sequences capable of specifically binding to each other is used for primer binding in, for example, northern or Southern blotting or PCR or RT-PCR.
Short oligonucleotides of the conserved region are advantageously used for hybridization. However, longer fragments of the nucleic acids of the invention or the complete sequences for hybridization may also be used. These standard conditions vary depending on the nucleic acid used (oligonucleotide, longer fragment or complete sequence) or depending on which type of nucleic acid (DNA or RNA) is used for hybridization. For example, the melting temperature of a DNA/DNA hybrid is about 10℃lower than the melting temperature of a DNA/RNA hybrid of the same length.
For example, standard conditions refer to temperatures between 42-58 ℃ in buffered aqueous solutions at concentrations between 0.1 and 5X SSC (1X SSC = 0.15m nacl,15mm sodium citrate, pH 7.2) or alternatively in the presence of 50% formamide, for example 5X SSC,50% formamide at 42 ℃. Advantageously, the hybridization conditions for the DNA to DNA hybrids are 0.1 XSSC at a temperature between about 20℃and 45℃and preferably between about 30℃and 45 ℃. For DNA: RNA hybrids, the hybridization conditions are advantageously 0.1 XSSC, at a temperature of about 30℃to 55℃and preferably about 45℃to 55 ℃. These described hybridization temperatures are examples of calculated melting temperature values for nucleic acids of about 100 nucleotides in length and 50% G+C content in the absence of formamide. The experimental conditions for DNA hybridization are described in the relevant genetics textbooks, e.g. Sambrook et al, 1989, and can be calculated using formulas known to the person skilled in the art, e.g. depending on the length of the nucleic acid, the type of hybrid or the g+c content. Further information on hybridization can be obtained by the person skilled in the art from the following textbooks: ausubel et al (edit), 1985,Current Protocols in Molecular Biology,John Wiley&Sons,New York; hames and Higgins (editions), 1985,Nucleic Acids Hybridization:A Practical Approach,IRL Press at Oxford University Press,Oxford; brown (edit), 1991,Essential Molecular Biology:A Practical Approach,IRL Press at Oxford University Press,Oxford.
"hybridization" can be carried out in particular under stringent conditions. Such hybridization conditions are described, for example, in Sambrook, J., fritsch, E.F., maniatis, T., in: molecular Cloning (A Laboratory Manual), 2 nd edition, cold Spring Harbor Laboratory Press, pages 1989,9.31-9.57 or Current Protocols in Molecular Biology, john Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
"stringent" hybridization conditions specifically denote: the filters were washed with 0.1 XSSC at 65℃after overnight incubation at 42℃in a solution consisting of 50% formamide, 5 XSSC (750 mM NaCl,75mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5 XDenhardt's solution, 10% dextran sulfate and 20. Mu.g/ml denatured sheared salmon sperm DNA.
The invention also relates to derivatives of the specifically disclosed or derivable nucleic acid sequences.
Thus, other nucleic acid sequences of the invention may be derived from the sequences specifically disclosed herein and may differ therefrom by addition, substitution, insertion or deletion of a single or several nucleotides and also encode polypeptides having the desired profile of properties.
Depending on the codon usage of the particular original or host organism, as well as naturally occurring variants, such as splice variants or allelic variants thereof, the invention also encompasses nucleic acid sequences which comprise so-called silent mutations or which have been altered compared to the specifically stated sequences.
It also relates to sequences obtainable by conservative nucleotide substitutions (i.e. substitution of the amino acid with an amino acid of the same charge, size, polarity and/or solubility).
The invention also relates to molecules derived from the specifically disclosed nucleic acids by sequence polymorphism. Due to natural variation, these genetic polymorphisms can exist among individuals within a population. These natural variations typically produce 1 to 5% variation in the nucleotide sequence of the gene.
Derivatives of the nucleic acid sequences according to the invention are, for example, allelic variants which have at least 60% homology, preferably at least 80% homology, very particularly preferably at least 90% homology over the entire sequence at the derived amino acid level (for homology at the amino acid level, reference should be made to the polypeptide details given above). Advantageously, the homology may be higher in a partial region of the sequence.
Furthermore, derivatives are also understood as meaning homologs of the nucleic acid sequences according to the invention, for example animal, plant, fungal or bacterial homologs, shortened sequences, single-stranded DNA or RNA of coding and noncoding DNA sequences. For example, the homologs have at the DNA level a homology of at least 40%, preferably at least 60%, particularly preferably at least 70%, very particularly preferably at least 80% over the entire DNA region of the sequences specifically disclosed herein.
Furthermore, derivatives are understood as meaning, for example, fusions with promoters. Promoters added to the nucleotide sequences may be modified by at least one nucleotide exchange, at least one insertion, inversion and/or deletion, without compromising the function or efficacy of the promoter. Furthermore, the efficacy of promoters may be increased by altering their sequence, or may be fully exchanged with promoters of more efficient, even different genus organisms.
5. Constructs of the invention
The invention also relates to expression constructs comprising a nucleotide sequence encoding a polypeptide or fusion protein of the invention under the genetic control of a regulatory nucleotide sequence; and vectors comprising at least one of these expression constructs.
According to the present invention, an "expression unit" refers to a nucleic acid having expression activity, which comprises a promoter as defined herein and which, upon functional binding to the nucleic acid or gene to be expressed, modulates expression, i.e. transcription and translation of the nucleic acid or the gene. Thus, in this case, it is also referred to as "regulatory nucleic acid sequence". In addition to the promoter, other regulatory elements, e.g., enhancers, may be present.
According to the invention, an "expression cassette" or "expression construct" refers to an expression unit which is functionally related to the nucleic acid to be expressed or the gene to be expressed. In contrast to expression units, expression cassettes thus comprise not only nucleic acid sequences which regulate transcription and translation, but also nucleic acid sequences which should be expressed as proteins as a result of transcription and translation.
In the context of the present invention, the term "expression" or "overexpression" describes the production or increase of the intracellular activity of one or more enzymes in a microorganism which are encoded by the corresponding DNA. For this purpose, it is possible, for example, to insert a gene in an organism, to replace an existing gene with another gene, to increase the copy number of a gene, to use a strong promoter or to use a gene encoding a corresponding enzyme having high activity, and these measures may optionally be combined.
Preferably, such constructs of the invention comprise a promoter 5 '-upstream and a terminator sequence 3' -downstream of each coding sequence, and optionally other conventional regulatory elements, in each case functionally related to the coding sequence.
According to the invention, a "promoter", "nucleic acid having promoter activity" or "promoter sequence" refers to a nucleic acid functionally related to the nucleic acid to be transcribed, which regulates the transcription of the nucleic acid.
In this context, a combination of "functionality" or "operability" refers to, for example, a sequential arrangement of the following elements: one of the nucleic acids having promoter activity and the nucleic acid sequence to be transcribed and optionally further regulatory elements, for example nucleic acid sequences capable of transcribing the nucleic acid and for example terminators, whereby each regulatory element can be made to function in the transcription of the nucleic acid sequence. This does not necessarily require direct bonding in a chemical sense. Genetic control sequences, such as enhancer sequences, may also exert their function on the target sequence from a more distant location or even from other DNA molecules. An arrangement is preferred in which the nucleic acid sequence to be transcribed is located behind (i.e. at the 3' -end of) the promoter sequence, so that the two sequences are covalently bound to each other. The distance between the promoter sequence and the nucleic acid sequence to be expressed transgenically may be less than 200bp (base pairs), or less than 100bp or less than 50bp.
Examples of other regulatory elements that may be mentioned, in addition to promoters and terminators, are targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, origins of replication, etc. Suitable regulatory sequences are described, for example, in Goeddel, gene Expression Technology: methods in Enzymology 185,Academic Press,San Diego,CA (1990).
The nucleic acid constructs of the invention comprise in particular sequences selected from those specifically mentioned herein or derivatives and homologues thereof, as well as nucleic acid sequences which may be derived from the amino acid sequences specifically mentioned herein, advantageously in operative or functional combination with one or more regulatory signals for controlling, e.g. increasing, gene expression.
In addition to these regulatory sequences, the natural regulation of these sequences may still be present in front of the actual structural gene and optionally may be genetically altered so that the natural regulation is turned off and the expression of the gene has been increased. The nucleic acid construct may also be of simpler design, i.e.without any additional regulatory signals being inserted in front of the coding sequence and without the native promoter being removed by its regulation. In contrast, the native regulatory sequences are silenced, so that no regulation occurs anymore and gene expression increases.
Preferred nucleic acid constructs also advantageously contain one or more of the above-described enhancer sequences, which are functionally associated with the promoter, allowing for increased expression of the nucleic acid sequence. Other advantageous sequences, such as other regulatory elements or terminators, may also be inserted at the 3' end of the DNA sequence. One or more copies of the nucleic acids of the invention may be included in a construct. The construct may also contain other markers, such as antibiotic resistance or auxotroph complementing genes, optionally for screening of the construct.
Examples of suitable regulatory sequences are contained in promoters such as cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacI q- 、T7-、T5-、T3-、gal-、trc-、ara-、rhaP(rhaP BAD )SP6-、λ-P R Or lambda-PL promoter, which is advantageously used in gram-negative bacteria. Other advantageous regulatory sequences are included, for example, in the gram-positive promoters ace, amy and SPO2, the yeast or fungal promoters ADC1, MF. Alpha., AC, P-60, CYC1, GAPDH, TEF, rp, ADH. Artificial promoters may also be used for regulation.
For expression, the nucleic acid construct is advantageously inserted into a vector of the host organism, for example a plasmid or a phage, which allows optimal expression of the gene in the host. In addition to plasmids and phages, vectors are understood to mean all other vectors known to the person skilled in the art, for example viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phages, cosmids and linear or circular DNA. These vectors may replicate autonomously in the host organism, or may replicate via a chromosome. These vectors represent another embodiment of the invention.
Suitable plasmids are, for example, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III in E.coli 113 -B1, λgt11 or pBdCI; pJAM2 in nocardia actinomycetes; pIJ101, pIJ364, pIJ702 or pIJ361 in Streptomyces; pUB110, pC194 or pBD214 in the bacillus; pSA77 or pAJ667 in coryneform bacteria; pALS1, pIL2 or pBB116 in fungi; 2 alpha M, pAG-1, YEp6, YEp13 or pEMBLYe23 in yeast or pLGV23, pGHlac in plants + pBIN19, pAK2004 or pDH51. The above plasmids represent a few choices of possible plasmids. Other plasmids are well known to those skilled in the art and can be found, for example, in Cloning Vectors (edited Pouwels P.H. et al Elsevier, amsterdam-New York-Oxford,1985,ISBN 0 444 904018).
In another embodiment of the vector, the vector containing the nucleic acid construct of the invention or the nucleic acid of the invention can advantageously be inserted into the microorganism in the form of linear DNA by heterologous or homologous recombination and integrated into the genome of the host organism. The linear DNA may comprise a linearization vector, such as a plasmid, or only a nucleic acid construct or a nucleic acid of the invention.
For optimal expression of heterologous genes in organisms, it is advantageous to alter the nucleic acid sequence using specific codon usage in the organism. Codon usage can be readily determined based on computer evaluation of other known genes of the organism.
The expression cassettes of the invention are produced based on fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator signal or polyadenylation signal. Common recombinant and cloning techniques are used for this, as described, for example, in T.Maniatis, E.F.Fritsch and J.Sambrook, molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory, cold Spring Harbor, NY (1989) and T.J.Silhavy, M.L.Berman and L.W.Enquist, experiments with Gene Fusions, cold Spring Harbor Laboratory, cold Spring Harbor, NY (1984) and Ausubel, F.M. et al, current Protocols in Molecular Biology, greene Publishing assoc.and Wiley Interscience (1987).
The recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector for expression in a suitable host organism to allow optimal expression of the gene in the host. Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. et al, public. Elsevier, amsterdam-New York-Oxford, 1985).
6. Hosts which can be used according to the invention
Depending on the context, the term "microorganism" refers to the starting microorganism (wild-type) or the genetically modified microorganism according to the invention, or both.
According to the invention, the term "wild-type" refers to the corresponding starting microorganism and does not necessarily need to correspond to a naturally occurring organism.
By means of the vectors of the invention, recombinant microorganisms can be produced which have been transformed, for example, with at least one vector of the invention and can be used for producing the polypeptides of the invention. Advantageously, the recombinant constructs of the invention described above are inserted into a suitable host system and expressed. Preferably, common cloning and transfection methods familiar to those skilled in the art are used, such as co-precipitation, protoplast fusion, electroporation, retroviral transfection, etc., to ensure expression of the nucleic acids in the respective expression systems. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F.Ausubel et al, public. Wiley Interscience, new York 1997, or Sambrook et al Molecular Cloning: A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y., 1989.
In general, all prokaryotes can be considered recombinant host organisms of the nucleic acid or nucleic acid construct of the invention. Bacteria are advantageously used as host organisms. Preferably, they are selected from natural or recombinant bacteria capable of producing PHA-, TAG-or WE-inclusion bodies, in particular the following genera for producing TAG: nocardia actinomycetes (nocardioform actinomycetes), in particular Rhodococcus (Rhodococcus), mycobacterium (mycrobacter), nocardia (Nocardia), gordonia (Gordonia), skeermania and Tsukamurella; streptomyces books (Streptomycetes) that produce TAG; acinetobacter (Acinetobacter) and Alcanivorax producing WE; and recombinant strains of the genus Escherichia (Escherichia), in particular Escherichia coli, corynebacterium (Corynebacterium), in particular Corynebacterium glutamicum (C.glutamicum) and Bacillus, in particular Bacillus subtilis.
The host organism according to the invention then preferably contains at least one nucleic acid sequence, nucleic acid construct or vector according to the invention which codes for an enzyme activity according to the definition given above.
The organisms used in the process of the invention are grown or cultivated in a manner familiar to the person skilled in the art, depending on the host organism. Typically, the microorganisms are grown in a liquid medium containing a carbon source, typically in the form of a sugar, a nitrogen source, typically an organic nitrogen source, such as a yeast extract or a salt, e.g. ammonium sulphate, trace elements such as iron, manganese and magnesium salts and optionally vitamins, at a temperature between 0 ℃ and 100 ℃, preferably between 10 ℃ and 60 ℃, and oxygen aeration. The pH of the liquid nutrient medium may be maintained at a fixed value, i.e. adjusted or not during growth. Growth may be performed batchwise, semi-batchwise or continuously. The nutrients may be provided at the beginning of the fermentation or may be subsequently provided semi-continuously or continuously.
7. Recombinant production of enzymes of the invention
The invention also relates to a method for producing an enzyme for use in the method of the invention by culturing a microorganism expressing said enzyme and isolating the desired product from the culture.
The microorganisms used according to the invention can be cultivated continuously or discontinuously in a batch process or in a fed-batch or repeated fed-batch process. Reviews of known culturing methods can be found in textbooks of Chmiel (bioprocessstechnik 1.einfu hrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, stuttgart, 1991)) or Storhas (Bioreaktoren und periphere Einrichtungen (viewaeg Verlag, braunschweig/Wiesbaden, 1994)).
The medium to be used must meet the requirements of the particular strain in an appropriate manner. Descriptions of culture media for various microorganisms are given in the american society of bacteriology handbook "general bacteriology methods handbook" (Washington d.c., USA, 1981).
These media which can be used according to the invention generally comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
Preferred carbon sources are sugars, such as monosaccharides, disaccharides or polysaccharides. Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar may also be added to the culture medium by complex compounds, such as molasses or other by-products from sugar refining. It may also be advantageous to add mixtures of various carbon sources. Other possible carbon sources are oils and fats, such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids, such as palmitic acid, stearic acid or linoleic acid, alcohols, such as glycerol, methanol or ethanol, organic acids, such as acetic acid or lactic acid.
The nitrogen source is typically an organic or inorganic nitrogen compound or a material containing such compounds. Examples of nitrogen sources include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids, or complex nitrogen sources, such as corn steep liquor, soybean meal, soybean protein, yeast extract, meat extract, and the like. The nitrogen sources may be used alone or as a mixture.
Inorganic salt compounds that may be present in the culture medium include chlorides, phosphates or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
Inorganic sulfur-containing compounds, such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, and organic sulfur compounds, such as mercaptans and mercaptans, can be used as sulfur sources.
Phosphoric acid, monopotassium phosphate or dipotassium phosphate or the corresponding sodium-containing salts can be used as phosphorus sources.
Chelating agents may be added to the medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydric phenols, such as catechol or protocatechuic acid, or organic acids, such as citric acid.
The fermentation media used in the present invention may also contain other growth factors such as vitamins or growth promoters including, for example, biotin, riboflavin, thiamine, folic acid, niacin, pantothenic acid, and pyridoxine. The growth factors and salts are typically derived from complex components of the culture medium, such as yeast extract, molasses, corn steep liquor, and the like. In addition, suitable precursors may be added to the medium. The exact composition of the compounds in the medium strongly depends on the particular experiment and has to be determined individually for each specific case. Information about medium optimisation can be found in textbooks "Applied microbiol. Physiolog, A Practical Approach" (Publ.P.M.Rhodes, P.F.Stanbury, IRL Press (1997) p.53-73,ISBN 0 19 963577 3). Growth media are also available from commercial suppliers such as standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
All components of the medium are sterilized by heating (20 minutes at 1.5 bar and 121 ℃) or by sterile filtration. The components may be sterilized together or, if desired, separately. All components of the medium may be present at the beginning of the growth, or optionally may be added continuously or by fed-batch.
The temperature of the culture is typically between 15 ℃ and 45 ℃, preferably between 25 ℃ and 40 ℃, and may be kept constant or may vary during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably about 7.0. The pH of the growth can be controlled during growth by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Defoamers, such as fatty acid polyglycol esters, can be used to control foaming. To maintain the stability of the plasmid, a suitable substance having a selective action (e.g., an antibiotic) may be added to the medium. Oxygen or an oxygen-containing gas mixture, such as ambient air, is fed into the culture to maintain aerobic conditions. The culture temperature is typically 20℃to 45 ℃. Cultivation is continued until a maximum amount of the desired product is formed. This is typically done in 10 hours to 160 hours.
Cells may be disrupted by high frequency ultrasound, by high pressure (e.g., in a French press), by osmosis, by the action of a detergent, lyase or an organic solvent, by a homogenizer, or by a combination of several methods as listed.
8. Reaction conditions of the biocatalytic process of the invention
The at least one enzyme present during the method of the invention or the single step of the multi-step method as defined above may be present in living cells, harvested cells, dead cells, permeabilized cells, crude cell extracts, purified extracts, or in substantially pure or fully pure form, which naturally or recombinantly produce the one or more enzymes. The at least one enzyme may be present in solution or as an enzyme immobilized on a carrier. One or several enzymes may be present in both soluble and immobilized form.
The process of the invention can be carried out in customary reactors known to the person skilled in the art and can be carried out on different scales, for example from laboratory scale (reaction volumes of a few ml to a few tens of liters) to industrial scale (reaction volumes of a few liters to a few thousands of cubic meters). If the lipase is used in a form encapsulated by non-living, optionally permeabilized cells, in the form of a more or less purified cell extract or in purified form, a chemical reactor can be used. The chemical reactor typically allows for control of the amount of the at least one enzyme, the amount of the at least one substrate, the pH, the temperature and the circulation of the reaction medium. When the at least one enzyme is present in a living cell, the process will be fermentation. In this case, the biocatalytic production will take place in a bioreactor (fermenter) in which the parameters necessary for the proper survival conditions of the living cells (e.g. nutrient-containing medium, temperature, aeration, the presence or absence of oxygen or other gases, antibiotics, etc.) can be controlled. Those skilled in the art are familiar with chemical or biological reactors, for example, chemical or biotechnological processes from laboratory scale to industrial scale, or processes for optimizing process parameters, which are also widely described in the literature (biotechnological processes see, for example, crueger und Crueger, biotechnologies-Lehrbuch der angewandten Mikrobiologie,2.Ed., r.oldbourg Verlag, munchen, wien, 1984).
Cells containing the at least one enzyme may be permeabilized by physical or mechanical means, such as ultrasound or radio frequency pulses, french press, or chemical methods, such as hypotonic media, lyase and detergent present in the media, or combinations of these methods. Examples of detergents are digitonin, n-dodecyl maltoside, octyl glycoside,X-100、20. Deoxycholate, CHAPS (3- [ (3-chloroacetyl propyl) dimethylammonium)]-1-propanesulfonate),P40 (ethylphenol poly (glycol ether)) and the like.
If the at least one enzyme is immobilized, it is attached to an inert carrier. Suitable carrier materials are known in the art and are disclosed, for example, in EP-A-1149849, EP-A-1 069 183 and DE-OS 100193773 and the references cited therein (all of which are specifically included in the context of carrier materials). Examples of suitable carrier materials are clays, clay minerals such as kaolinite, diatomaceous earth, perlite, silica, alumina, sodium carbonate, calcium carbonate, cellulose powders, anion exchange materials, synthetic polymers such as polystyrene, acrylic resins, phenolic resins, polyurethanes and polyolefins such as polyethylene and polypropylene. For the preparation of the carrier-bound enzyme, the carrier material is generally used in the form of a fine powder, with porous forms being preferred. The particle size of the support material is generally not more than 5mm, in particular 2mm. Where the at least one enzyme is present in a whole cell preparation, the whole cell preparation may be present in free or immobilized form. Suitable carrier materials are, for example, calcium alginate or carrageenan. The enzyme and the cell may be directly linked by glutaraldehyde. A variety of immobilization methods are known in the art (e.g., J.Lande and A.Margolin, immobilization of Enzymes "in K.Drauz und H.Waldmann, enzyme Catalysis in Organic Synthesis 2002, vol. III,991-1032, wiley-VCH, weinheim).
The conversion reaction may be carried out batchwise, semi-batchwise or continuously. The reactants (and optionally nutrients) may be provided at the beginning of the reaction or may be subsequently provided semi-continuously or continuously.
The reactions of the present invention may be carried out in aqueous or non-aqueous reaction media, depending on the particular type of reaction. The ester cleavage reaction is preferably carried out in the presence of water, in particular in the presence of a water-organic solvent system, preferably a two-phase system. The esterification reaction may be carried out in advance in the absence of water, more particularly in the presence of an organic solvent which is free or substantially free of water.
The aqueous medium may contain a suitable buffer to adjust the pH to a value of 5 to 9, such as 6 to 8.
The nonaqueous medium may be substantially free of water, i.e., contain less than about 1% or 0.5% by weight water.
In particular, the biocatalytic process is carried out in an organic nonaqueous medium. As suitable organic solvents, there may be mentioned, for example, aliphatic hydrocarbons of 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane; aromatic carbohydrates, such as benzene, toluene, xylene, chlorobenzene or dichlorobenzene, aliphatic acyclic compounds and ethers, such as diethyl ether, methyl-tert-butyl ether, ethyl-tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether; or a mixture thereof. Preferably, an organic solvent is used that has the ability to form a biphasic solvent system with water.
The concentration of reactants/substrates can be adjusted to the optimal reaction conditions, which may depend on the particular enzyme used. For example, the initial substrate concentration may be 0.1 to 0.5M, e.g., 10 to 100mM.
The reaction temperature may be adjusted to the optimal reaction conditions, which may depend on the specific enzyme used. For example, the reaction may be carried out at a temperature of from 0 to 70 ℃, for example from 20 to 50 or from 25 to 40 ℃. Examples of reaction temperatures are about 30 ℃, about 35 ℃, about 37 ℃, about 40 ℃, about 45 ℃, about 50 ℃, about 55 ℃, and about 60 ℃.
The process may be run until equilibrium between substrate and product is achieved, but may be stopped earlier. Typical treatment times are from 1 minute to 25 hours, in particular from 10 minutes to 6 hours, for example from 1 hour to 4 hours, in particular from 1.5 hours to 3.5 hours.
8.1 Selective enzymatic esterification of 3-unsaturated Carboxylic acids such as Gao Fani acid
In a preferred embodiment, a mixture of (3E/Z) isomers of 3-unsaturated carboxylic acids, in particular (3E, 7E) -and (3Z, 7E) -Gao Fani acids, aliphatic alcohols and optionally organic solvents, is treated with a lipase to effect the esterification reaction.
Non-limiting examples of suitable lipases are candida antarctica (Candida antarctica) lipase (CALB), and immobilized analogues thereof, such as Novozym
Non-limiting examples of suitable alcohols are aliphatic C 1 -C 20 Alcohols, such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, n-pentanol, n-hexanol, n-heptanol and n-octanol.
Non-limiting examples of suitable solvents are in particular aliphatic hydrocarbons, such as hexane, cyclohexane, heptane, octane; aromatic hydrocarbons such as toluene, xylene; dialkyl ethers such as MTBE and diisopropyl ether.
In a preferred embodiment, one equivalent of alcohol is used per equivalent of (3E) -carboxylic acid, in particular the (3E, 7E) -isomer of Gao Fani acid, to obtain substantially complete conversion. The use of smaller amounts of alcohol limits the yield of esters.
The reaction is suitably carried out at a temperature in the range of about 0 ℃ to +80℃. The reaction progress may be controlled by GC or HPLC analysis.
A separable acid/ester mixture of the carboxylic acid isomers is obtained.
8.2 Selective enzymatic saponification of 3-unsaturated Carboxylic esters, e.g. Gao Fani acid esters
In another embodiment, separable mixtures of isomers are obtained by enzymatic ester cleavage using a 3E/Z-isomer mixture of a 3-unsaturated carboxylic acid, particularly a mixture of (3E, 7E) -and (3Z, 7E) -isomers of an alkyl Gao Fani acid ester. In the reaction, free 3E-acids, in particular the (3E, 7E) -Gao Fani acid isomers, and unreacted 3Z esters, in particular unreacted (3Z, 7E) -Gao Fani acid esters, are obtained.
In a preferred embodiment, the isomeric mixture of alkyl carboxylates, optionally dissolved in an organic solvent, is converted by application of a lipase in the presence of water.
Non-limiting examples of suitable lipases are candida antarctica (Candida antarctica) lipase (CALB), and immobilized analogues thereof, such as Novozym
Non-limiting examples of suitable alkyl groups for esters are: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and sec-butyl.
Non-limiting examples of suitable solvents are aliphatic hydrocarbons such as hexane, cyclohexane, heptane; aromatic hydrocarbons such as toluene and xylene; ethers, for example MTBE and diisopropyl ether, THF.
8.3 preparation of (-) -ambrox
Preparation can be carried out starting from stereoisomerically pure (3E/7E) homofarnesic acid obtained by the invention by applying a known process as described in scheme 1 above.
The disclosures of EP16156410 and WO2010/139719 and WO2012/066059, as described in scheme 1, which are incorporated herein by reference, describe biocatalytic conversion of unsaturated substrates by the use of cyclases of different origins.
Sclareolide, e.g. obtained by catalytic conversion of the (3E/7E) -Gao Fani acid of the invention by cyclase, followed by chemical reduction (e.g. by LiAlH 4 Or NaBH 4 ) To form ambrox-1, 4-diol [ Mookherjee et al; perfumer and Flavourist (1990), 15:27]. Ambrox-1, 4-diol can then be chemically converted to (-) -ambrox by different methods (see, e.g., US 5,274,134).
The biocatalytic synthesis of the compound (-) -ambroxol is also described in the literature [ Neumann et al; biol Chem Hoppe Seyler (1986), 367:723]. The molecule is obtained from homofarnesol ((3Z, 7E) -4,8, 12-trimethyltridec-3, 7, 11-trien-1-ol) and squalene-cereal grain cyclase (SHC) from alicyclobacillus acidocaldarius (Alicyclobacillus acidocaldarius) (formerly Bacillus acidocaldarius (Bacillus acidocaldarius)) as catalyst.
9. Product separation
The process of the present invention may further comprise the step of recovering the end product or intermediate product, optionally in substantially pure form as a stereoisomer or enantiomer. The term "recovering" includes extracting, harvesting, separating or purifying the compound from the culture or reaction medium. Recovery of the compounds may be performed according to any conventional separation or purification method known in the art, including, but not limited to, treatment with conventional resins (e.g., anion or cation exchange resins, nonionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, alumina, etc.), changing pH, solvent extraction (e.g., with conventional solvents such as ethanol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.
The identity and purity of the isolated product can be determined by known techniques such as High Performance Liquid Chromatography (HPLC), gas Chromatography (GC), spectroscopic techniques (e.g., IR, UV, NMR), staining methods, TLC, NIRS, enzymes or microbiological assays. ( See, for example: patek et al (1994) appl. Environ. Microbiol.60:133-140; malakhova et al (1996) Biotekhnologica 1127-32; UNd Schmidt et al (1998) Bioprocess Engineer.19:67-70.Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd.A27, VCH:Weinheim, S.89-90, S.521-540, S.540-547, S.559-566,575-581und S.581-587; michal, G (1999) Biochemical Pathways: an Atlas of Biochemistry and Molecular Biology, john Wiley and Sons; fallon, A.et al (1987) Applications of HPLC in Biochemistry in: laboratory Techniques in Biochemistry and Molecular Biology, bd.17. )
The following examples are given solely for the purpose of illustration of the invention. Many possible variations that will be apparent to those skilled in the art are also within the scope of the invention.
Examples section:
A. material
(1) Enzymes
Lipase: novozym 435; commercial products of Novozymes;
immobilized candida antarctica (Candida antarctica) lipase B
Amino acid sequence:
Cyclase: zm-SHC-1 see below
Unless otherwise indicated, recombinant proteins are cloned and expressed by standard methods, e.g., as described in Sambrook, j., fritsch, e.f. and manitis, t., molecular cloning: A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory, cold Spring Harbor Laboratory Press, cold Spring Harbor, NY, 1989.
(2) Chemical substance
Mixtures of isomers of high farnesic acid are obtained, for example, by the method described in european patent application No. EP 17157950.1 filed 2/24/2017.
Isomer mixtures of homofarnesoic acid esters have been prepared from isomer mixtures of homofarnesoic acid (e.g., according to the generally known acid-catalyzed esterification of carboxylic acids with alcohols (so-called Fischer esterification-Chemische Berichte 28,1895,3252-3258).
All other chemicals used were laboratory grade.
A.The method comprises the following steps:
1. HPLC parameters for analysis of high farnesic acid compounds
Device Agilent Series 1100
Column Chiralpak AD-RH 5 μm 150 x 4,6mm von
Eluent: -A water, containing 0.1% by volume of H 3 PO 4
Acetonitrile containing 0.1% by volume of H 3 PO 4
Time (minutes) | %B | Flow rate |
0,0 | 30 | 1,2 |
25,0 | 70 | 1,2 |
30,0 | 100 | 1,2 |
30,1 | 30 | 1,2 |
Detector UV-detector λ=205 nm, bw=5 nm
Flow rate 1,2ml/min
Injection 5. Mu.L
The temperature is 40 DEG C
Duration of 35min
Pressure of about 70bar
High farnesic acid | Esters of | Retention time [ min] |
3Z,7E | - | 10,31 |
3E,7E | - | 11,73 |
3Z,7E | Methyl ester | 17,33 |
3E,7E | Methyl ester | 19,37 |
3Z,7E | Ethyl ester | 18,87 |
3E,7E | Ethyl ester | 21,28 |
3Z,7E | Isopropyl ester | 20,06 |
3E,7E | Isopropyl ester | 21,73 |
3Z,7E | Butyl ester | 23,25 |
3E,7E | Butyl ester | 26,24 |
3Z,7E | Octyl ester | 29,77 |
3E,7E | Octyl ester | 30,88 |
2. GC parameters for sclareolide analysis
The conversion of homofarnesic acid to sclareolide can be determined by the following GC system:
column 10m Optima 1
Temperature profile:
0min:100℃
5 ℃/min to 200 DEG C
After 5min
30 ℃/min to 320 DEG C
Then constant
The method has a duration of 30min
The temperature of the injector is 280 DEG C
Retention Time (RT):
high farnesic acid, peak 1 at 11.7min, peak 2 at 12.1min;
sclareolide about 13.5min
A calibration series was established using a reliable material (Sigma, cat# 358002) by means of which the concentration of unknown samples was determined.
2. Lipase Activity assay
The tributyrin test was performed according to Beisson, F.et al Eur.J.Lipid Sci.Technol.2000, 133-153.
B. Examples:
reference example 1 cloning and expression in E.coli of Zm-SHC-1
The gene for the cyclase can be amplified from Zymomonas mobilis (Zymomonas mobilis) by means of the oligonucleotides Zm-SHC_fw and Zm-SHC_rev.
In each case, 100ng of primers Zm-SHC_fw and Zm-SHC_rev were mixed in equimolar ratios. PCR was performed using genomic DNA from zymomonas mobilis (ATCC 31821), following the manufacturer's instructions, using Pwo-polymerase (Roche Applied Science) and the following temperature gradient procedure: 3 minutes at 95 ℃; 30 cycles were performed: at 95℃for 30 seconds, at 50℃for 30 seconds, at 72℃for 3 minutes; 10 minutes at 72 ℃; until use at 4 ℃. The PCR products (. About.2.2 kb) were separated by agarose gel electrophoresis (1.2% electrophoresis gel, invitrogen) and column chromatography (GFX kit, amersham Pharmacia), followed by sequencing (sequencing primers: zm-SHC_fw and Zm-SHC_rev). The obtained sequence matches the published sequence.
The PCR product was digested with restriction endonucleases NdeI and BamHI and ligated into the appropriately digested vector pDHE19.2 [9]. The obtained plasmid was sequenced to obtain the nucleic acid sequence shown in SEQ ID No. 1. The corresponding amino acid sequence is shown below/(SEQ ID NO: 2):
/>
/>
the plasmid pDHE-Zm-SHC-1 was transformed into E.coli strain TG10 pAgro4 pHSG575 [ Takeshita et al, gene 1987,61:63-74; tomoyasu et al, mol Microbiol 2001,40:397-413]. The recombinant E.coli was designated E.coli LU15568.
Reference example 2 providing recombinant high farnesyl cyclase from Zymomonas mobilis
From a suitable 2ml preculture, E.coli LU15568 was inoculated at 37℃in 20ml LB-Amp/Spec/Cm (100. Mu.g/l ampicillin; 100. Mu.g/l spectinomycin; 20. Mu.g/l chloramphenicol), 0.1mM IPTG, 0.5g/l rhamnose in a 100ml conical flask (with baffles) for 16 hours, centrifuged at 5000. Mu.g/10 min and stored at 4 ℃. Protein extracts were prepared by suspending the cell pellet in 15ml of disruption buffer (0.2M Tris/HCl,0.5M EDTA,pH 8.0), 375U benzonase (e.g., novagen, 25U/. Mu.L), 40. Mu.L PMSF (100 mM in isopropanol), 0.8g sucrose and about 0.5 mg lysozyme. The reaction mixture was mixed and incubated on ice for 30 minutes. Thereafter, the mixture was frozen at-20 ℃.
After the reaction mixture was thawed, it was made up to about 40ml with distilled water and incubated on ice for an additional 30 minutes.
Thereafter, ultrasound (HTU-Soni 130, from g.heineman,amplitude 80%15 "pulse/15" pause) the cells were destroyed 3 times for 3 minutes. After disruption, cell debris was removed by centrifugation at 26 x 900 g for 60 minutes at 4 ℃. The supernatant was discarded, and the pellet was resuspended in 100ml of solubilization buffer (50 mM Tris/HCl,10mM MgCl2x6H2O,1%Triton X-100, pH 8.0) and crushed in a Potter for about 5 minutes. Thereafter, the suspension was kept on ice for 30 minutes.
The homogenized extract was centrifuged at 26900×g for 1 hour at 4 ℃ and the pellet was discarded. The extract is used in enzyme assays and can be stored at-20 ℃ for several weeks without suffering from activity loss. The protein content was in the range of 1 mg/ml.
Reference example 3 Activity determination of recombinant cyclase from E.coli LU15568
Homofarnesoic acid ((3E, 7E) -4,8, 12-trimethyltridec-3, 7, 11-trienoic acid) was incubated with the protein preparation described in reference example 2. Specifically, 0.0412g of homofarnesic acid was weighed (20 mM; purity 85.1% in the reaction mixture, consisting of Z, Z0.44%, E, Z10.13%, E74.93%) and 2.913ml of water was removed; 0.350ml sodium citrate buffer (1M sodium citrate pH 5.4), 0.560ml MgCl 2 (0.5M solution) and the mixture was stirred at 37℃for 30 minutes. Coli LU15568 homogenate (protein content 35 mg/ml) was added to start the reaction and warmed to 37 ℃. The reaction mixture was stirred on a magnetic stirrer in an oil bath at ph5.0 at 37 ℃ for 24 hours at maximum stirring speed. The pH was adjusted during the reaction using 0.5M HCl. After 24 hours incubation, the incubation was performed by using 1000ml of n-heptane/n-propanol 3:2 vortex for 30 seconds, 0.500ml was extracted from the reaction mixture. The organic supernatant after phase separation was used for GC analysis (see fig. 1).
Using the analysis described in more detail below, 74.5% conversion was determined from the total 82.7% of the E, E isomer.
EXAMPLE 1 preparation of (3E, 7E) -Gao Fani butyl acrylate
71g (283 mmol) of a mixture of (3E, 7E) -Gao Fani acid and (3Z, 7E) -Gao Fani acid in a molar ratio of 56:44 are dissolved in 360ml of n-heptane. 21g (283 mmol) of n-butanol and 470mg Novozym 435 are added. The mixture was stirred at 23℃for 48 hours. The enzyme was isolated by filtration. 115ml of methanol and 5ml of water are added at a temperature of 0 ℃. The pH of the mixture was adjusted to ph=12 by adding aqueous sodium hydroxide (25%) with stirring at a temperature <10 ℃.
The stirrer was stopped and the lower phase was separated. After removal of the solvent 43.5g (119 mmol) of high-farnesoid butyl ester are obtained, which have a (3E, 7E) -content of >97%.
Example 2 influence of different alcohols on enzymatic esterification selectivity of high farnesic acid in absence of solvent:
2g (8 mmol) of a 57:43 mixture of (3E, 7E) -Gao Fani acid and (3Z, 7E) -Gao Fani acid are dissolved in 15ml of different alcohols and stirred at 23℃in the presence of 20mg of Novozym 435. At specific time intervals, the composition of the reaction mixture was analyzed by HPLC. The results are summarized in the following table 1.
Table 1:
example 3: influence of combinations of different alcohols and the solvent heptane on the enzymatic esterification selectivity of high farnesic acid
2g (8 mmol) of a 57:43 mixture of (3E, 7E) -Gao Fani acid and (3Z, 7E) -Gao Fani acid are dissolved in 15ml of heptane. 8mmol of the different alcohols and 20mg of Novozym 435 were added and the mixture was stirred at 23 ℃. At predetermined time intervals, the composition of the reaction mixture was analyzed by HPLC. The results are shown in Table 2.
Table 2:
example 4: influence of butanol and combinations of different solvents on the selectivity of enzymatic esterification of high farnesic acid
2g (8 mmol) of a 57:43 mixture of (3E, 7E) -Gao Fani acid and (3Z, 7E) -Gao Fani acid are dissolved in 15ml of different solvents. 8mmol of butanol and 20mg of Novozym 435 were added and the mixture was stirred at 23 ℃. At predetermined time intervals, the composition of the reaction mixture was analyzed by HPLC. The results are shown in Table 3.
Table 3:
example 5: preparation of free (3E, 7E) -Gao Fani acids by enzymatic ester cleavage
2g (7.56 mmol) of methyl (3E, 7E) -and (3Z, 7E) -Gao Fani acid esters ((3E, 7E): ratio (3Z, 7E) =51:49) were dissolved in 50ml toluene. 10ml of water and 50mg of Novozym 435 were added. The mixture was stirred at 23 ℃. After 6 hours, the composition of the reaction mixture was analyzed as follows:
36% (3E, 7E) -Gao Fani acid methyl ester,
49% (3Z, 7E) -Gao Fani acid methyl ester,
15% (3E, 7E) -Gao Fani acid, and
<0.1% (3 z,7 e) -Gao Fani acid.
The enzyme was removed by filtration and the reaction mixture was adjusted to pH >9 with sodium carbonate. The aqueous lower phase was separated. The pH of the aqueous phase was adjusted to a value <4 with acid (10% hydrochloric acid). The phase was then extracted with toluene. The toluene phase obtained contains more than 95% of pure (3E, 7E) -Gao Fani acid.
Sequence:
nucleic acid/amino acid sequences of 1-326 SHC genes of SEQ ID NO
327-328PCR primer
Nucleic acid/amino acid sequence of SEQ ID NO 329,330 Lipase CALB
References cited herein are expressly incorporated herein by reference.
Claims (20)
1. A process for separating the 3- (E) -isomer of an unsaturated carboxylic acid compound of the general formula (I) from an isomer mixture comprising the 3- (E) -and 3- (Z) -isomers of the carboxylic acid compound, wherein the isomer mixture is subjected to an enzymatic conversion reaction catalyzed by a lipase (EC 3.1.1.3) which preferentially converts the 3- (E) -isomer and the conversion product of the 3- (E) -isomer is separated from the reaction mixture,
Wherein the method comprises the steps of
R 1 Is H or straight-chain or branched, saturated or unsaturated C 1 -C 20 A hydrocarbyl group;
R 3 is H or C 1 -C 4 -a hydrocarbon group;
R 2 c being linear or branched, saturated or unsaturated 1 -C 20 -a hydrocarbon group;
provided that if R 3 Is C 1 -C 4 -a hydrocarbon group, R 2 Represents a hydrocarbon group containing at least one additional carbon atom,
wherein the lipase is from a Candida sp.
2. The process of claim 1, wherein the conversion reaction comprises an enzymatic esterification of an acid of formula (Ia);
wherein the method comprises the steps of
R 2 And R is 3 As defined above;
and wherein predominantly 3- (E) -esters are formed.
3. The method of claim 1, wherein the conversion reaction comprises an enzymatic ester cleavage reaction of an ester of formula (Ib);
wherein the method comprises the steps of
R 1 Is straight chain or branched chain,Saturated or unsaturated C 1 -C 20 -, in particular C 4 -C 20 -a hydrocarbon group;
and R is 2 And R is 3 As defined above;
and wherein predominantly 3- (E) -acid is formed.
4. The process of claim 1, wherein the conversion reaction is carried out in an organic solvent or a water-organic solvent.
5. The process of claim 1, wherein the carboxylic acid compound is a 3- (E)/7- (E) -Gao Fani acid compound of formula (II)
Wherein R is 1 As defined above.
6. A process for preparing an unsaturated 3- (E) -carboxylic acid of the general formula (Ia):
wherein the method comprises the steps of
R 2 And R is 3 As defined above;
wherein the method comprises the steps of
a) Combining the isomer mixture comprising the 3- (E) -and 3- (Z) -isomers of the carboxylic acid of formula (Ia) in the presence of a compound of formula R 1 Enzymatic esterification in the presence of an alkanol of OH, wherein R is 1 C being linear or branched, saturated or unsaturated 1 -C 20 -a hydrocarbon group; and in the presence of a lipase as defined in claim 1;
b) Isolating the 3- (E) -carboxylate formed in step a), and
c) Saponifying the isolated ester of step b) to the corresponding 3- (E) -carboxylic acid of formula (Ia).
7. A process for preparing an unsaturated 3- (E) -carboxylic acid of the general formula (Ia):
wherein the method comprises the steps of
R 2 And R is 3 As defined above;
wherein the method comprises the steps of
a) A mixture of isomers comprising the 3- (E) -and 3- (Z) -isomers of the carboxylic acid esters of formula (Ib) is subjected to an enzymatic ester cleavage in the presence of a lipase as defined in claim 1,
wherein the method comprises the steps of
R 1 C being linear or branched, saturated or unsaturated 1 -C 20 -a hydrocarbon group; and is also provided with
R 2 And R is 3 As defined above;
b) Isolating said 3- (E) -carboxylic acid formed in step a).
8. A process as claimed in claim 6 or 7, wherein an organic solvent as defined in claim 4 is used.
9. The method of any one of claims 1, 6 and 7, wherein the 3- (E) -isomer of the unsaturated carboxylic acid is 3- (E)/7- (E) -Gao Fani acid.
10. The method of any one of claims 1, 6 and 7, wherein the isomer mixture comprises a mixture of 3- (E)/7- (E) -Gao Fani acid and 3- (Z)/7- (E) -Gao Fani acid; or R is 1 Mixtures of 3- (E)/7- (E) -Gao Fani acid esters and 3- (Z)/7- (E) -Gao Fani acid esters of alkanols of OH, wherein R 1 C being linear or branched, saturated or unsaturated 1 -C 20 -hydrocarbyl groupsA group.
11. A process for preparing 3- (E)/7- (E) -Gao Fani acid as claimed in claim 10,
wherein the method comprises the steps of
a) The isomer mixture comprising 3- (E)/7- (E) -Gao Fani acid and 3- (Z)/7- (E) -Gao Fani acid is represented by formula R 1 Enzymatic esterification in the presence of an alkanol of OH, wherein R is 1 C being linear or branched, saturated or unsaturated 1 -C 20 -a hydrocarbon group; and in the presence of a lipase as defined in claim 1 in a solvent as defined in claim 4;
b) Separating the 3- (E)/7- (E) -Gao Fani acid ester formed in step a) from unreacted acid, and
c) The isolated 3- (E)/7- (E) -Gao Fani acid ester was saponified to 3- (E)/7- (E) -Gao Fani acid.
12. A process for preparing 3- (E)/7- (E) -Gao Fani acid as claimed in claim 10,
Wherein the method comprises the steps of
a) Subjecting an isomer mixture comprising 3- (E)/7- (E) -Gao Fani acid ester and 3- (Z)/7- (E) -Gao Fani acid ester to an enzymatic ester cleavage in the presence of a lipase as defined in claim 1 in a solvent as defined in claim 4; and is also provided with
b) Separating the 3- (E)/7- (E) -Gao Fani acid formed in step a) from the unreacted ester.
13. The process according to claim 12, wherein the solvent as defined in claim 4 in step a) is a water-organic solvent.
14. The method of any one of claims 11-13, wherein the separation in step b) is by distillation.
15. The method of any one of claims 11-13, wherein the separation in step b) is by extraction.
16. Preparation method of (-) -ambrox of formula (III)
The method comprises the following steps:
a) Obtaining the 3- (E)/7- (E) -Gao Fani acid by applying the method as defined in any one of claims 1-13;
b) Acid reduction of 3- (E)/7- (E) -Gao Fani to 3- (E)/7- (E) -Gao Fani alcohol and
c) The 3- (E)/7- (E) -Gao Fani alcohol is enzymatically cyclized to (-) -ambrox.
17. Preparation method of (-) -ambrox of formula (III)
The method comprises the following steps:
a) Obtaining the 3- (E)/7- (E) -Gao Fani acid by applying the method as defined in any one of claims 1-13;
b) Enzymatic cyclization of 3- (E)/7- (E) -Gao Fani acid to sclareolide of formula (IV),
and is also provided with
c) Chemically converting the sclareolide to (-) -ambroxol.
18. The method as claimed in claim 16 or 17, wherein the enzymatic cyclization is carried out in the presence of an intramolecular transferase (e.c. 5.4).
19. The method of claim 18, wherein the intramolecular transferase (e.c. 5.4) is a squalene-graminene cyclase (e.c. 5.4.99.17) that exhibits high farnesyl cyclase activity.
20. The method of claim 19, wherein the cyclase is from zymomonas mobilis (Zymomonas mobilis) comprising the amino acid sequence of SEQ ID No. 2 or an amino acid sequence having at least 60% sequence identity to SEQ ID No. 2.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP17157974 | 2017-02-24 | ||
EP17157974.1 | 2017-02-24 | ||
PCT/EP2018/054502 WO2018154048A1 (en) | 2017-02-24 | 2018-02-23 | Method for the preparation of (3e,7e)-homofarnesic acid or (3e,7e)-homofarnesic acid ester |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110325646A CN110325646A (en) | 2019-10-11 |
CN110325646B true CN110325646B (en) | 2024-02-09 |
Family
ID=58227907
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201880013734.XA Active CN110325646B (en) | 2017-02-24 | 2018-02-23 | Process for preparing (3E, 7E) -Gao Fani acid or (3E, 7E) -Gao Fani acid esters |
Country Status (7)
Country | Link |
---|---|
US (1) | US11136611B2 (en) |
EP (1) | EP3585902B1 (en) |
JP (1) | JP7263244B2 (en) |
CN (1) | CN110325646B (en) |
BR (1) | BR112019015594A2 (en) |
ES (1) | ES2890814T3 (en) |
WO (1) | WO2018154048A1 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114989000B (en) * | 2017-02-24 | 2024-05-10 | 巴斯夫欧洲公司 | Process for preparing unsaturated carboxylic acids by carbonylation of allyl alcohol and its acylated products |
MX2020008509A (en) | 2018-02-13 | 2020-09-25 | Basf Se | Method for the isomerization of a 3-(z)-unsaturated carboxylic acid to the 3-(e)-isomer. |
CN110540979B (en) * | 2019-09-07 | 2021-02-02 | 华南理工大学 | Hydrogen peroxide tolerant lipase mutant and application thereof |
CN110468117B (en) * | 2019-09-07 | 2021-03-09 | 华南理工大学 | Organic solvent tolerant lipase mutant and application thereof |
GB202011823D0 (en) | 2020-07-30 | 2020-09-16 | Givaudan Sa | Method |
CN112029684B (en) * | 2020-09-02 | 2022-03-22 | 浙江工业大学 | Mineralized bacteria suitable for underwater engineering concrete repair and application thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102695695A (en) * | 2009-12-18 | 2012-09-26 | 奇华顿股份有限公司 | Organic compounds |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3345375A1 (en) | 1983-12-15 | 1985-06-27 | Basf Ag, 6700 Ludwigshafen | METHOD FOR PRODUCING 4-SUBSTITUTED BUT-3-EN-L-CARBONIC ACIDS AND THEIR ESTERS |
DE3912318A1 (en) | 1989-04-14 | 1990-10-18 | Henkel Kgaa | METHOD FOR THE STEREOSELECTIVE PRODUCTION OF 8 (ALPHA), 12-OXIDO-13,14,15,16-TETRANORLABDANE |
DE19931847A1 (en) | 1999-07-09 | 2001-01-11 | Basf Ag | Immobilized lipase |
DE10019380A1 (en) | 2000-04-19 | 2001-10-25 | Basf Ag | Process for the production of covalently bound biologically active substances on polyurethane foams and use of the supported polyurethane foams for chiral syntheses |
DE10019377A1 (en) | 2000-04-19 | 2001-10-25 | Basf Ag | Immobilization of biologically active substances, especially enzymes, comprises contacting a foamed polymeric material with the substance |
US8759043B2 (en) | 2009-06-05 | 2014-06-24 | Basf Se | Biocatalytic production of ambroxan |
WO2012066059A2 (en) | 2010-11-17 | 2012-05-24 | Basf Se | Method for the biocatalytic cyclisation of terpenes and cyclase mutants which can be used in said method |
US8932839B2 (en) | 2010-11-17 | 2015-01-13 | Basf Se | Method for the biocatalytic cyclization of terpenes and cyclase mutants employable therein |
EP2922813B1 (en) * | 2012-11-23 | 2016-10-12 | Firmenich SA | Hydrocarbonylation or methoxycarbonylation of 1,3-diene derivatives with palladium complex |
GB201507207D0 (en) * | 2015-04-24 | 2015-06-10 | Givaudan Sa | Enzymes and applications thereof |
MX2018010024A (en) | 2016-02-19 | 2018-11-09 | Basf Se | Enzymatic cyclization of homofarnesylic acid. |
CN114989000B (en) | 2017-02-24 | 2024-05-10 | 巴斯夫欧洲公司 | Process for preparing unsaturated carboxylic acids by carbonylation of allyl alcohol and its acylated products |
-
2018
- 2018-02-23 WO PCT/EP2018/054502 patent/WO2018154048A1/en active Search and Examination
- 2018-02-23 ES ES18706520T patent/ES2890814T3/en active Active
- 2018-02-23 US US16/487,978 patent/US11136611B2/en active Active
- 2018-02-23 JP JP2019545916A patent/JP7263244B2/en active Active
- 2018-02-23 EP EP18706520.6A patent/EP3585902B1/en active Active
- 2018-02-23 CN CN201880013734.XA patent/CN110325646B/en active Active
- 2018-02-23 BR BR112019015594-5A patent/BR112019015594A2/en unknown
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102695695A (en) * | 2009-12-18 | 2012-09-26 | 奇华顿股份有限公司 | Organic compounds |
Non-Patent Citations (3)
Title |
---|
Cyclization of E,E-homofarnesic acid and its related compounds;Saito Akira et al;《Chemistry Letters》;19841231;第591-594页 * |
STN数据库 99734-74-4;STN数据库;《STN数据库》;19860118;第1页 * |
许建和.脂肪酶.《生物催化工程》.2008, * |
Also Published As
Publication number | Publication date |
---|---|
WO2018154048A1 (en) | 2018-08-30 |
CN110325646A (en) | 2019-10-11 |
JP7263244B2 (en) | 2023-04-24 |
US11136611B2 (en) | 2021-10-05 |
JP2020508062A (en) | 2020-03-19 |
BR112019015594A2 (en) | 2020-05-26 |
EP3585902B1 (en) | 2021-07-14 |
EP3585902A1 (en) | 2020-01-01 |
US20200123586A1 (en) | 2020-04-23 |
ES2890814T3 (en) | 2022-01-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110325646B (en) | Process for preparing (3E, 7E) -Gao Fani acid or (3E, 7E) -Gao Fani acid esters | |
JP6989513B2 (en) | Enzymatic cyclization of homofarnesyl acid | |
JP5101510B2 (en) | Novel dehydrogenase, derivative thereof and method for producing optically active alkanol | |
US8715970B2 (en) | Enzymatically catalyzed method of preparing mono-acylated polyols | |
US8951750B2 (en) | Malonate decarboxylases for industrial applications | |
WO2009103547A1 (en) | Process for the production of gamma-aminobutyric acid | |
JP2021519579A (en) | How to make vanillin | |
EP2459727A2 (en) | Biocatalyst for catalytic hydroamination | |
US20060211099A1 (en) | L-carnitin dehydrogenases, their derivatives and method for producing substituted (s) alkanols | |
JP7467440B2 (en) | Lipoxygenase-catalyzed production of unsaturated C10 aldehydes from polyunsaturated fatty acids (PUFAs) | |
JP5820778B2 (en) | Protein with esterase activity | |
RU2333958C2 (en) | Butinol 1 esterase | |
JP7507739B2 (en) | Enzymatic cyclization of homofarnesylic acid. | |
EP2280075A1 (en) | Biocatalyst for catalytic hydroamination |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |